Compositions and methods comprising klk3, psca, or folh1 antigen

[0001] The invention provides recombinant KLK3 peptides, recombinant nucleotide molecules encoding them, recombinant Listeria strains containing them, and their immunogenic and therapeutic uses.

BACKGROUND OF THE INVENTION [0002] Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host's immune system. Bacterial antigens such as Salmonella enterica and Mycobacterium bovis BCG remain on the phagosome and stimulate CD4 T cells⁺ via antigen presentation by Class II major histocompatibility molecules. In contrast, bacterial antigens such as Listeria monocytogenes leave the phagosome to the cytoplasm. Phagolisosomal escape of L. monocytogenes is a unique mechanism that facilitates the antigenic presentation of Listeria antigens via the major class I histocompatibility system. This escape depends on the thiol-forming pore of cytolysin activated listeriolysin O (LLO).

[0003] ActA is a surface-associated Listeria protein, and acts as a scaffold in infected host cells, facilitating the polymerization, assembly and activation of host actin polymers, for the movement of Listeria through cytoplasm. Shortly after entering the cytosol of a mammalian cell L. monocytogenes induces polymerization of host actin filaments and uses the force generated by actin polymerization to displace first intracellularly and then from cell to cell. The single bacterial protein, ActA, is responsible for mediating actin nucleation and actin-based motor. The ActA protein provides multiple binding sites for host cytoskeleton components, thereby acting as a scaffold for assembling cellular machinery for actin polymerization. The NH2 end of ActA binds to monomeric actin and acts as a constitutively active nucleation stimulating factor, by stimulating the actin nucleation activity. ActA and hly, both, are members of the 10-kb gene cluster, regulated by PrfA transcription activator, and are up-regulated by approximately 226-fold in mammalian cytosol.

[0004] Prostate cancer is the most common type of cancer in male Americans and it is the second most important cause of cancer-related death in this population. The prostate specific antigen (PSA) is a marker of prostate cancer that is highly expressed by prostate cancers. There is a long-felt need to develop compositions and methods for increasing the immunogenicity of antigens, particularly antigens useful in the prevention and treatment of cancers, and intracellular pathogens.

BRIEF DESCRIPTION OF THE INVENTION [0005] The invention provides a recombinant Listeria strain expressing a fusion peptide - a kallikrein-related peptidase 3 (KLK3) peptide that is operably linked to a N-terminal non-hemolytic listeriolysin peptide (LLO), said fusion peptide containing the SEQ ID sequence 54 or a sequence at least 99% homologous thereto, wherein said N-terminal LLO peptide increases the immunogenicity of the fusion peptide, and wherein the KLK3 peptide does not contain a signal sequence. In an embodiment, the recombinant Listeria strain is passaged by an animal host.

[0006] The recombinant Listeria strain may be an auxotrophic Listeria strain or a recombinant Listeria monocytogenes strain. The auxotrophic Listeria strain may be a dal / dat mutant or may additionally include a deletion in the endogenous ActA gene. The auxotrophic Listeria strain may contain an episomal expression vector containing a metabolic enzyme that complements the auxotrophic Listeria, and the metabolic enzyme may be an alanine racemase enzyme or a Daminacid transferase enzyme.

[0007] The invention also provides a recombinant polypeptide comprising a KLK3 peptide fusion peptide that is operably linked to an N-terminal LLO peptide, said recombinant polypeptide having the sequence SEQ ID NO: 54 or a sequence at least 99% homologous thereto, said N-terminal LLO peptide increases the immunogenicity of the fusion peptide, and wherein the KLK3 peptide does not contain a signal sequence. The invention also provides a nucleotide molecule encoding a recombinant polypeptide, and a recombinant vaccine vector encoding a recombinant polypeptide or comprising a nucleotide molecule encoding a recombinant polypeptide.

[0008] The invention also provides a vaccine comprising a recombinant Listeria strain, a recombinant polypeptide or nucleotide molecule encoding a recombinant polypeptide and an adjuvant.

[0009] The invention provides a recombinant Listeria strain, or immunogenic composition comprising a recombinant polypeptide, or a recombinant nucleotide, for use as a medicine.

[0010] The invention further provides a recombinant Listeria strain, or immunogenic composition comprising a recombinant polypeptide, or a recombinant nucleotide, for use in inducing an anti-KLK3 immune response in a subject.

[0011] The invention also provides a recombinant Listeria strain, recombinant polypeptide, or recombinant nucleotide, for use in the treatment, inhibition or suppression of a protein-expressing prostate cancer associated with kallikrein peptidase 3 (KLK3) in a subject by which the subject triggers an immune response against the expressing KLK3 protein for prostate cancer.

[0012] The recombinant polypeptide may be obtained by a method comprising the step of chemically coupling a polypeptide comprising KLK3 peptide to a polypeptide comprising an N-terminal LLO peptide.

BRIEF DESCRIPTION OF THE DRAWINGS [0013]

Figure 1. Lm-E7 and Lm-LLO-E7 use different systems for expression, expression and secretion of E7. Lm-E7 was generated by inserting the gene cassette into the orfZ domain of the L. monocytogenes genome (A). The hly promoter directs the expression of the hly signal sequence, and the first five amino acids (AA) of LLO are followed by HPV-16 E7. B), Lm-LLO-E7 was generated by transforming the prfA strain^- XFL-7 plasmid pGG-55. pGG-55 has the hly promoter directing expression of the nonhemolytic LLO-E7 fusion. pGG-55 also contains the prfA gene for selection for XFL-7 plasmid preservation in vivo.

Figure 2. Lm-E7 and Lm-LLO-E7 secrete E7. Lm-Gag (lane 1), Lm-E7 (lane 2), Lm-LLO-NP (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane 5) and 10403S (lane 6) were cultured overnight at 37 ° C in Luria-Bertoni broth. Equivalent bacterial numbers, as determined by absorbance - OD at 600 nm - were collected and 18 ml of each supernatant was TCA precipitated. E7 expression was analyzed by Western hybridization. The hybridized filter was treated with an anti-E7 mAb probe followed by HRP conjugated anti-mouse antibody (Amersham) and then developed using ECL detection reagents.

Figure 3. A. Immunotherapeutic efficacy of LLO-E7 fusion against cancer.

Tumor size in millimeters in mice is shown 7, 14, 21, 28 and 56 days after tumor inoculation. Naive mice: unfilled triangles; Lm-LLO-E7: filled wheels; Lm-E7: filled diamonds; Lm-Gag: X's; and Lm-LLO-NP: + 3 signs. B. Immunotherapeutic efficacy of LLO-Ova fusion against cancer.

Figure 4. Splenocytes from Lm-LLO-E7-immunized mice proliferate upon exposure to TC-1 cells. C57BL / 6 mice were immunized and boosted with Lm-LLO-E7, Lm-E7, or rLm control strains. Splenocytes were harvested 6 days after boost, and placed in dishes with exposed TC-1 cells as shown in proportions. Cells were pulsed³H thymidine and collected. Cpm was defined as (experimental cpm) (no-TC-1 control).

Figure 5. Immunotherapeutic efficacy of NP antigen against LM-expressed NP tumor. Tumor size in millimeters in mice is shown 10, 17, 24 and 38 days after tumor inoculation. Naive: X's; mice treated with Lm-LLO-NP: filled diamonds; Lm-NP: squares; Lm-Gag: filled wheels.

-4 Figure 6. Presentation of vaccinia virus constructs expressing various forms of the HPV16 E7 protein.

Figure 7. VacLLOE7 causes long-term regression of 2 × 10 established tumors⁵ TC-1 cells injected sc [subcutaneously] to C57BL / 6 mice. Mice were injected 11 and 18 days after tumor challenge 10⁷ PFU VacLLOE7, VacSigE7LAMP-1 or VacE7 / mouse ip [intraperitoneal] or left untreated (naive). Eight mice were used per treatment group, and the cross-section of each tumor (mean of two measurements) is shown for the indicated days after tumor inoculation.

Figure 8. A. Schematic representation of the plasmid inserts used to form 4 LM vaccines. Insert Lm-LLO-E7 contains all the Listeria genes used. It contains the hly promoter, the first 1.3 kb of the hly gene (which encodes the LLO protein), and the HPV-16 E7 gene. The first 1.3 kb hly contains the signal sequence (ss) and the PEST region. Lm-PESTE7 contains the hly promoter, signal sequence, and PEST and E7 sequences, but no remaining portion of the truncated LLO gene. And .m-APHST-H7 lacks the PEST region, but contains the hly promoter, signal sequence, E7, and the remainder of the truncated LLO. Lm-E7epi only has the hly promoter, signal sequence and E7. B. Schematic representation of the pActA-E7 expression system used for E7 expression and secretion from recombinant Listeria bacteria. The hly promoter (pHLY) directs expression, the prfA gene is used to select plasmid behavior by recombinant Listeria in vivo. C. Top panel: Listeria constructs containing the PEST region induce tumor regression. Filled triangles: naive mice; wheels: Lm-LLO-E7; squares: LmE7epi; + signs: Lm-APEST-PA; unfilled triangles: Lm-PEST-E7. D. Average tumor size on day 28 after tumor challenge in two separate experiments. E. Listeria constructs containing PEST regions induce a higher percentage of E7-specific lymphocytes in the spleen. Mean and SE [standard error] are shown for the data from three experiments.

Figure 9. Tumor size in mice treated with Lm-ActA-E7 (unfilled squares), Lm-E7 (filled circles), Lm-LLO-E7 (X), and naive mice (unvaccinated; filled triangles).

Figure 10. A. Induction of E7-specific IFN-gamma secreting CD8 T cells⁺in spleens and the number of penetrating tumors in mice treated with TC-1 tumor cells and then administered Lm-E7, Lm-LLO-E7, Lm-ActA-E7, or no vaccine (naive). B. Induction and penetration of E7-specific CD8 + cells in the spleens and mouse tumors described for (A).

Figure 11. Listeria constructs containing PEST regions induce a higher percentage of E7-specific lymphocytes within the tumor. A. Representative data from one experiment. B. Mean and SE for data from all three experiments.

Figure 12. Schematic map of pAdv34 in pGG55. CAT (G-), chloramphenicol acetyltransferase for E. coli; CAT (G +), chloramphenicol acetyltransferase for Lm;

-5Ori, replication [initiation] region; prfA, Lm factor A regulating pathogenicity; LLOPSA, the fusion between the gene encoding the C-terminal truncated listeriolysin O, including its promoter, and the gene encoding human PSA.

Figure 13. Expression and secretion of LLO-PSA fusion protein by Lm-LLOPSA. Bacteria were grown overnight and cell supernatants were precipitated in the presence of 10% TCA at 4 ° C. Proteins were separated on SDS-PAGE and hybridized with anti-PSA (A) and anti-LLO (B) antibodies. The PSA positive control was PSA / Vaccinia infected BscI cell lysate. The parent bacterial strain XFL7 and two unrelated Lm strains expressing fragments of the human Her2 / neu antigen were used as negative controls.

Figure 14. Lm-LLO-PSA uptake and growth in macrophages. Mouse macrophage-like cells (J774A.1) were infected in vitro with MOI [ang. multiplicity of infection; infection efficiency] of order 1: 1 with Lm-LLO-PSA, native Lm XFL7 or Lm 10403s wild-type strains. Intracellular growth was monitored for 8 hours, taking duplicate samples every other hour from the cells, followed by titration of the bacteria in BHI dishes. CFU: colony forming units.

Figure 15. Tumor regression after Lm-LLO-PSA immunization. Groups of 8 mice were inoculated sc 5 x 10⁶ T-PSA23 cells on day 0 and immunized three times, with one week interval, ip Lm-LLO-PSA, Lm-LLO-E7 or no (naive) immunization was used. Tumors were monitored weekly using an electronic caliper and expressed as the average of two perpendicular diameters. Mice were sacrificed when the tumors reached 15-18 mm in diameter. The results were presented as the tumor size of each individual mouse. The table shows the number of mice in each group that were tumor-free (upper panel) or survived (lower panel) relative to the total number of mice per group, 1 week or 7-8 weeks after tumor inoculation. This experiment was repeated three times with similar results.

Figure 16. PSA-specific CD8 + T cells in spleens and tumors. T-PSA23 cells were implanted sc as a mixture with matrigel. Mice were immunized twice with Lm-LLO-PSA or Lm-LLO-E7 or left naive. Spleens and tumors were extracted 6 days after the second immunization and tested for the presence of a CD8 positive T cell tetramer⁺/ PSA using FACS analysis. Immunizations with Lm-LLO-PSA resulted in the generation of PSA-specific CD8 T cells⁺ both in spleen (upper panel) and cancer (lower panel). The numbers in each corner indicate the% of PSA positive cells relative to the total number of CD8 T cells⁺ in every tissue.

Figure 17. Effect of vaccination with Lm vaccines on the presence of Treg in spleens and tumors. Isolated splenocytes and TIL extracted from 3 tumor-bearing mice from the previous experiment were pooled and stained for CD4, CD25 and FoxP3 to explain the effect of Lm-LLO-PSA immunization on the presence of

-6 Training in these tissues. Each column presents% CD25 T cell population⁺/ FoxP3⁺ relative to the total number of CD4 T cells⁺ in each pool.

Figure 18. IFN-γ secretion stimulated by vaccination / u-LLO-PSA detected in the ELISpot assay. Mice were immunized three times with 0.1 LD50 LmLLO-PSA. Isolated splenocytes were prepared and incubated overnight in the presence of 1 μΜ of PSA specific H2D peptide^b, HCIRNKSVIL. IFN-γ secretion by isolated cells was detected using the ELISpot kit from Mabtech and expressed as point forming cells (SFC) / million cells. Splenocytes from Lm-LLO-WT1 immunized or naive mice were used as negative controls. (* indicates statistical significance for naive groups at p <0.0001).

Figure 19. IFN-γ production stimulated by vaccination / u-LLO-PSA detected by intracellular staining. Mice were immunized three times with 0.1 1D50 Lm-11O-PSA. Isolated splenocytes were prepared and incubated for 5 hours in the presence of PSA specific H2D peptide^b. IFN-γ production by activated CD8 T cells⁺ determined as described in the methods. Splenocytes from Lm-LLO-WT1 immunized or naive mice were used as negative controls.

Figure 20. PSA-specific cytotoxic responses in mice. Animals were immunized three times with 0.1 1D50 1m-11O-PSA, Lm-11O-HPV16E7E6 (as Lm negative control) or left naive. Isolated splenocytes were prepared and incubated with PSA-expressing stimulator cells for 5 days. CT1 [cytotoxic T lymphocyte] assay was performed for 4 hours by incubating effector cells (E) with E14 target cells pulsed with PSA H2D peptide^b: A) at different E: T ratios / at a constant peptide concentration (1 μΜ); or B) at a constant E: T ratio of 1:25 but with different concentrations of peptide.

Figure 21. Comparison of the anti-tumor activity of Lm-LLO-PSA with other PSA-based vaccines. Groups of 8 mice were inoculated sc 5 x 10⁶ TPSA23 cells on day 0 and immunized twice, with one week interval, LmLLO-PSA, vaccinia-PSA or pDNA (PSA + GM-CSF) or no immunization (naive). Tumors were monitored weekly using an electronic caliper and expressed as the average of two perpendicular diameters. Mice were sacrificed when the tumors reached 20 mm in diameter. Results are presented as tumor size in each individual mouse (A). The table shows the number of mice in each group that were tumor free or survived for 8 weeks after tumor inoculation. B) Average tumor sizes in each group. Mice were sacrificed when the tumors reached 20 mm in diameter. They were assigned a value of 20 mm to get an average curve. This experiment was repeated twice with similar results.

Figure 22. Lm-PSA stability. Lm-PSA was cultured and passaged for 7 consecutive days in vitro. Plasmid DNA was purified from bacterial samples

Taken each day during in vitro growth and tested by amplification of the PSA gene by PCR (A) or EcoRI / HindIII restriction mapping of the plasmid (B).

DETAILED DESCRIPTION OF THE INVENTION [0014] The invention provides KLK3-LLO fusion peptides, recombinant polypeptides containing them, recombinant nucleotide molecules encoding them, recombinant Listeria strains containing them, and using immunogenic and therapeutic methods.

[0015] More specifically, the invention provides a recombinant Listeria strain expressing a fusion peptide - kallikrein-related peptidase 3 (KLK3) peptide that is operably linked to the N-terminal nonhemolytic listeriolysin peptide (LLO), said fusion peptide containing the sequence SEQ ID NO: 54 or a sequence at least 99% homologous thereto, wherein said N-terminal LLO peptide increases the immunogenicity of the fusion peptide, and wherein the KLK3 peptide does not contain a signal sequence. In an embodiment, the recombinant Listeria strain may have been passaged by an animal host.

[0016] The recombinant Listeria strain may be an auxotrophic Listeria strain or a recombinant Listeria monocytogenes strain. The auxotrophic Listeria strain may be a dal / dat mutant or may additionally include a deletion in the endogenous ActA gene. The auxotrophic Listeria strain may contain an episomal expression vector containing a metabolic enzyme that complements the auxotrophic Listeria, and the metabolic enzyme may be an alanine racemase enzyme or a Daminacid transferase enzyme.

[0017] The invention also provides a recombinant polypeptide comprising a KLK3 peptide fusion peptide that is operably linked to an N-terminal LLO peptide, said recombinant polypeptide having the sequence SEQ ID NO: 54 or a sequence at least 99% homologous thereto, said N-terminal LLO peptide increases the immunogenicity of the fusion peptide, and wherein the KLK3 peptide does not contain a signal sequence. The invention also provides a nucleotide molecule encoding a recombinant polypeptide, and a recombinant vaccine vector encoding a recombinant polypeptide or comprising a nucleotide molecule encoding a recombinant polypeptide.

[0018] The invention also provides a vaccine comprising a recombinant Listeria strain, a recombinant polypeptide or nucleotide molecule encoding a recombinant polypeptide, and an adjuvant.

[0019] The invention provides a recombinant Listeria strain, or immunogenic composition comprising a recombinant polypeptide, or a recombinant nucleotide, for use as a medicine.

[0020] The invention further provides a recombinant Listeria strain, or immunogenic composition comprising a recombinant polypeptide or recombinant nucleotide, for use in inducing an anti-KLK3 immune response in a subject.

[0021] The invention also provides a recombinant Listeria strain, recombinant polypeptide, or recombinant nucleotide, for use in treating, inhibiting or suppressing prostate cancer expressing a protein associated with kallikrein peptidase 3 (KLK3) in a subject by which the subject triggers an immune response against the expressing protein KLK3 prostate cancer.

[0022] The recombinant polypeptide may be obtained by a method comprising the step of chemically coupling a KLK3 peptide-containing polypeptide to a polypeptide comprising an N-terminal LLO peptide.

[0023] In an embodiment, the invention provides a recombinant Listeria strain expressing a kallikrein-related peptidase 3 (KLK3) -LLO-related fusion peptide, said fusion peptide comprising the sequence SEQ ID NO: 54 or a sequence at least 99% homologous thereto. In another embodiment, the KLK3 peptide sequence in the fusion protein is selected from SEQ ID NO: 25, 32, 34, and 39. In another embodiment, the KLK3 peptide sequence comprises the sequence selected from SEQ ID No: 25, 32, 34, and 39 . In another embodiment, the KLK3 peptide is an immunogenic fragment of a larger KLK3 peptide, wherein the larger KLK3 peptide sequence is a sequence selected from SEQ ID No: 25, 32, 34 and 39. In another embodiment, the KLK3 peptide sequence comprises an immunogenic fragment of a sequence selected from SEQ ID No: 25, 32, 34 and 39.

[0024] "KLK3 peptide" is lacking in KLK3 signal peptide. In another embodiment, the term refers to a KLK3 protein that contains the entire KLK3 sequence except for the KLK3 signal peptide. "KLK3 signal sequence" refers, in another embodiment, to any signal sequence found in nature at the KLK3 protein. In another embodiment, the KLK3 protein of the methods and compositions of the invention does not contain any signal sequence.

[0025] In one embodiment, the "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments of the organism or tissue) that is different from the majority of the population, but is still sufficiently similar to the common type to be considered one of them , for example, splice variants.

[0026] In one embodiment, the "isoform" refers to the version of the molecule, for example a protein, with only slight differences compared to another isoform or version of the same protein. In one embodiment, the isoforms may be produced from different but related genes, or in another embodiment, they may be from the same gene, but after alternative splicing. In another embodiment, the isoforms are the result of single nucleotide polymorphisms.

[0027] In one embodiment, the "fragment" refers to a protein or polypeptide that is shorter or contains fewer amino acids than a full-length protein or polypeptide. In another embodiment, the fragment relates to a nucleic acid that is shorter or contains

-9 fewer nucleotides than full-length nucleic acid. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment is an intra-sequential stretch of protein, peptide or nucleic acid. In one embodiment, the fragment is a functional fragment. In another embodiment, the fragment is an immunogenic fragment. In one embodiment, the fragment has 10-250 nucleic acids or amino acids.

[0028] In one embodiment, the "homologue" refers to a nucleic acid or amino acid sequence that shares a certain percentage of sequence identity with a particular nucleic acid or amino acid sequence. In another embodiment, a sequence useful in the composition and methods as provided herein can be the homologue of any of the sequences described herein. In an embodiment, the homologue has at least 99% identity to the specified sequence. In one embodiment, it is to be understood that the homologue of any of the sequences, as provided herein and / or as described herein, is considered to be part of the invention.

[0029] In one embodiment, "heterologous" as described herein describes a nucleic acid, amino acid, peptide, polypeptide, protein etc. derived from a species other than the reference species. Thus, for example, a Listeria strain expressing a heterologous polypeptide, in one embodiment will express a polypeptide that is not native or endogenous to the Listeria strain, or in another embodiment a polypeptide that is not normally expressed by the Listeria strain, or in another embodiment sources other than the Listeria strain.

[0030] In one embodiment, the "endogenous" described herein describes a nucleic acid, amino acid, peptide, polypeptide, protein, etc. that has been developed or which originates from a reference organism, or is due to causes involving the reference organism. In another embodiment, endogenous is native.

[0031] In another embodiment, kallikrein-related peptidase 3 (KLK3 protein), which is a source of KLK3 peptide from the methods and compositions of the invention, is a PSA protein.

[0032] In another embodiment, the KLK3 protein is mature KLK3 protein. In another embodiment, the KLK3 protein is a KLK3 pro-protein. In another embodiment, the leader sequence has been removed from the mature KLK3 protein from the methods and compositions of the invention. An example of a mature KLK3 is encoded by aa378-1088 with SEQ ID No: 40.

[0033] In another embodiment, the KLK3 protein, which is the source of the KLK3 peptide from the methods and compositions of the invention, is human KLK3 protein.

[0034] In another embodiment, the KLK3 protein has the sequence SEQ ID No: 25 (GenBank accession number X14810). In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 25.

[0035] In another embodiment, KLK3 is encoded by a nucleotide molecule with the sequence SEQ ID No: 26 (GenBank accession number X14810). In another embodiment, KLK3 is encoded by residues 401-446, 1688-1847, 3477-3763, 3907-4043, 5413-5568 or a combination thereof, with SEQ ID No: 26. In another embodiment, KLK3 is encoded by the homologue of SEQ ID No: 26. In another embodiment, KLK3 is encoded by the variant SEQ ID No: 26. In another embodiment, KLK3 is encoded by the SEQ ID No: 26 isoform. In another embodiment, KLK3 is encoded by a fragment of SEQ ID No: 26.

[0036] In another embodiment, the KLK3 protein has the sequence SEQ ID No: 32 (GenBank accession number NM_001648). In another embodiment, the KLK3 protein is a fragment from SEQ ID No: 32.

[0037] In another embodiment, KLK3 is encoded by a nucleotide molecule having the sequence SEQ ID No: 33 (GenBank accession number NM_001648). In another embodiment, KLK3 is encoded by residues 42-827 of SEQ ID No: 33. In another embodiment, KLK3 is encoded by homologue SEQ ID No: 33. In another embodiment, KLK3 is encoded by variant SEQ ID No: 33. In another embodiment, KLK3 is encoded by the isoform of SEQ ID No: 33. In another embodiment, KLK3 is encoded by the fragment SEQ ID No: 33.

[0038] In another embodiment, the KLK3 protein has the sequence SEQ ID No: 34 (GenBank accession number BC056665). In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 34.

[0039] In another embodiment, KLK3 is encoded by a nucleotide molecule having the sequence SEQ ID No: 35 (GenBank accession number BC056665). In another embodiment, KLK3 is encoded by residues 47-832 of SEQ ID No: 35. In another embodiment, KLK3 is encoded by the homologue of SEQ ID No: 35. In another embodiment, KLK3 is encoded by the variant SEQ ID No: 35. In another embodiment, KLK3 is encoded by the isoform of SEQ ID No: 35. In another embodiment, KLK3 is encoded by the fragment SEQ ID No: 35.

[0040] In another embodiment, the KLK3 protein has the sequence SEQ ID No: 39 (GenBank accession number AJ459783). In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 39.

[0041] In another embodiment, KLK3 is encoded by a nucleotide molecule having the sequence SEQ ID No: 40 (GenBank accession number X07730). In another embodiment, KLK3 is encoded by residues 67-1088 of SEQ ID No: 40. In another embodiment, KLK3 is encoded by homologue SEQ ID No: 40. In another embodiment, KLK3 is encoded by variant SEQ ID No: 40. another embodiment, KLK3 is encoded by the isoform of SEQ ID No: 40. In another embodiment, KLK3 is encoded by the fragment SEQ ID No: 40.

[0042] In another embodiment, the KLK3 protein has the sequence given in one of the following GenBank accession numbers: BC005307, AJ310938, AJ310937, AF335478,

-11AF335477, M27274 and M26663. In another embodiment, the KLK3 protein is encoded by the sequence given in one of the above GenBank accession numbers.

[0043] In another embodiment, the KLK3 protein is encoded by the sequence given in one of the above GenBank accession numbers: NM_001030050, NM_001030049, NM_001030048, AJ459782, AJ512346 or AJ459784.

[0044] In another embodiment, the KLK3 protein has a sequence that comprises the sequence given in one of the following GenBank accession numbers: X13943, X13942, X13940, X13941 and X13944.

[0045] In another embodiment, the invention provides a vaccine comprising the recombinant Listeria strain of the invention and an adjuvant.

[0046] In another embodiment, the invention provides an immunogenic composition comprising the recombinant Listeria strain of the invention.

[0047] In another embodiment, the recombinant Listeria strain expresses a recombinant polypeptide that comprises a KLK3: LLO fusion peptide. In another embodiment, the recombinant Listeria strain comprises a recombinant polypeptide, wherein the recombinant peptide comprises a KLK3: LLO fusion peptide. In another embodiment, the recombinant Listeria strain comprises a recombinant nucleotide encoding the recombinant polypeptide.

[0048] KLK3: LLO fusion peptide expressed by recombinant Listeria increases the immunogenicity of KLK3 peptide.

[0049] The non-KLK3 / non-FOLH1 peptide in the fusion peptide of the invention is an LLO peptide, which in another embodiment may be a nonhemolytic LLO peptide.

[0050] In another embodiment, the invention provides a recombinant Listeria strain comprising the recombinant polypeptide of the invention. In another embodiment, the invention provides a recombinant Listeria strain comprising a recombinant nucleotide encoding a recombinant polypeptide of the invention. In another embodiment, the Listeria vaccine strain is a strain of the species Listeria monocytogenes (LM). In another embodiment, the invention provides a composition comprising a Listeria strain. In another embodiment, the invention provides an immunogenic composition comprising a Listeria strain.

[0051] In another embodiment, the Listeria strain is a recombinant Listeria seeligeri strain. In another embodiment, the Listeria strain is a recombinant Listeria grayi strain. In another embodiment, the Listeria strain is a recombinant Listeria ivanovii strain. In another embodiment, the Listeria strain is a recombinant Listeria murrayi strain. In another embodiment, the Listeria strain is a recombinant Listeria welshimeri strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art.

[0052] In another embodiment, the recombinant Listeria strain of the invention is passaged through an animal host. Passaging can maximize the effectiveness of the strain as a vaccine vector, stabilize the immunogenicity of the strain

Listeria, stabilize the virulence of the Listeria strain, increase the immunogenicity of the Listeria strain, increase the virulence of the Listeria strain, remove unstable sub-strains of the Listeria strain, and / or reduce the incidence of unstable sub-strains of the Listeria strain. In another embodiment, the Listeria strain comprises a genomic insertion of the gene encoding the KLK3 peptide. In another embodiment, the Listeria strain carries a plasmid containing the gene encoding the recombinant KLK3: LLO fusion peptide. Methods of passaging a recombinant Listeria strain in an animal host are well known in the art and are described, for example, in US Patent Application Publication No. 2006/0233835. In another embodiment, the passage is carried out by any other method known in the art.

[0053] In another embodiment, the recombinant Listeria strain used in the methods of the invention was stored in a frozen cell bank or a lyophilized cell bank. A cell bank may be a major cell bank, a working cell bank and / or a good manufacturing practice (GMP) cell bank. The cell bank may be for the production of clinical grade material and / or in accordance with regulatory practices for human use.

[0054] "Good manufacturing practices" are defined, in another embodiment, in (21 CFR 210-211) United States Code of Federal Regulations. In another embodiment, "good manufacturing practices" are defined by means of other standards regarding the production of material with a clinical grade of purity or for human consumption; e.g. a country standard other than the United States.

[0055] In another embodiment, the recombinant Listeria strain used in the invention is from a vaccine dose lot or from a frozen base material or lyophilized base material prepared by the method disclosed herein.

[0056] In another embodiment, the cell bank, frozen stock material or batch of vaccine doses of the invention have a viability after thawing greater than 90%. In another embodiment, thawing occurs after storage for cryopreservation or storage in a frozen state for 24 hours. In another embodiment, the storage is 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, or storage is 1 year.

[0057] In another embodiment, the cell bank, frozen stock material or batch of vaccine doses of the invention are cryopreserved by a method that includes culturing the Listeria strain culture in nutrient media, freezing the culture in a solution containing glycerol, and storing the Listeria strain at a temperature below - 20 degrees Celsius. In another embodiment, the temperature is about -70 degrees Celsius. In another embodiment, the temperature is about -70-80 degrees Celsius.

[0058] In another embodiment, the cell bank, frozen stock material or batch of vaccine doses of the invention is subjected to cryopreservation by a method that includes culturing the Listeria strain culture in a defined medium (as described

-13 below), freezing the culture in a solution containing glycerol and storing the Listeria strain at a temperature below -20 degrees Celsius. In another embodiment, the temperature is about -70 degrees Celsius. In another embodiment, the temperature is about -70-80 degrees Celsius. In another embodiment, any specified microbial media can be used in this method.

[0059] In another embodiment, the culture (e.g., culture of the Listeria vaccine strain that is used to produce a batch of the Listeria vaccine strain) is inoculated from a cell bank. In another embodiment, the culture is inoculated from frozen basic material. In another embodiment, the culture is inoculated from the starter culture. In another embodiment, the culture is inoculated from a colony. In another embodiment, the culture is inoculated in a mean logarithmic growth phase. In another embodiment, the culture is inoculated at approximately a mean logarithmic growth phase. In another embodiment, the culture is inoculated in a different growth phase.

[0060] The freezing solution may contain another colligative additive or additive with antifreeze properties instead of or in addition to glycerol. In another embodiment, the additive is mannitol, DMSO, sucrose or any other colligative antifreeze additive or additive that is known in the art.

[0061] The nutrient medium used to culture the Listeria strain culture may be LB, TB, or a modified animal-free Terrific Broth broth. In another embodiment, the nutrient medium is a defined medium. In another embodiment, the nutrient medium is a defined medium according to the invention. In another embodiment, the nutrient medium is any other type of nutrient medium known in the art.

[0062] The growth step may be carried out in a shake flask, such as in a shake flask, in a batch fermenter, in a stirred tank or flask, in an air-operated fermenter, in a batch reactor, in a continuous reactor cells or in an immobilized cell reactor.

[0063] In another embodiment, the pH is maintained constant during growth of the culture (e.g., in a batch fermenter). In another embodiment, the pH is maintained on the order of about 7.0, about 6, about 6.5, about 7.5, or the pH is about 8. In another embodiment, the pH is

6.5-7.5. In another embodiment, the pH is 6-8. In another embodiment, the pH is 6-7. In another embodiment, the pH is 7-8.

[0064] In another embodiment, the temperature is kept constant during the growth of the culture. In another embodiment, the temperature is maintained at about 37 ° C. In another embodiment, the temperature is 37 ° C, 25 ° C, 27 ° C, 30 ° C, 34 ° C, 36 ° C, 38 ° C or in another embodiment, the temperature is 39 ° C.

[0065] In another embodiment, the dissolved oxygen concentration is maintained constant during growth of the culture. In another embodiment, the dissolved oxygen concentration is maintained at 20% saturation. In another embodiment, the concentration means 15% saturation, or 16% saturation, or 18%, or 22%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%,

-14 or 55%, 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 100% saturation, or almost 100% saturation.

[0066] In another embodiment, the Listeria culture is immediately frozen in liquid nitrogen and then stored at the final freezing temperature. In another embodiment, the culture is frozen more gradually; e.g. by placing the culture vial at final storage temperature.

[0067] In another embodiment, the culture storage temperature is between 20 and -80 degrees Celsius (° C). In another embodiment, the temperature is well below -20 ° C. In another embodiment, the temperature is no more than -70 ° C, or is -70 ° C, or is about -70 ° C. In another embodiment, the temperature is -20 ° C or about -20 ° C, -30 ° C, -40 ° C, -50 ° C, -60 ° C, -80 ° C, -30 - -70 ° C, -40 - -70 ° C, -50 -70 ° C, -60 - -70 ° C, -30 - -80 ° C, -40 to -80 ° C, -50 - -80 ° C, -60 - -80 ° C, -70 - -80 ° C, or the temperature is less than -70 ° C or less than -80 ° C.

[0068] Methods for lyophilization and cryopreservation of recombinant Listeria strains are well known to those skilled in the art.

[0069] A composition comprising Listeria means, in another embodiment, an immunogenic composition. In another embodiment, the composition is immunogenic in nature, by the feature of containing the Listeria strain of the invention. In another embodiment, the composition further comprises an adjuvant.

[0070] In some embodiments, the term "comprising" refers to the inclusion of other recombinant polypeptides, amino acid sequences or nucleic acid sequences, as well as the inclusion of other polypeptides, amino acid sequences, or nucleic acid sequences that may be known in the art, in one form embodiments may comprise antigens or polypeptides, amino acid sequences or Listeria nucleic acid sequences. In some embodiments, the term "consisting essentially of" refers to a composition for use in methods as provided herein that has a specific recombinant polypeptide, amino acid sequence, or nucleic acid sequence, or fragment thereof. However, other polypeptides, amino acid sequences, or nucleic acid sequences that are not directly involved in the utility of the recombinant polypeptides may be included. In some embodiments, the term "consisting of" refers to a composition for use in methods as provided herein having a specific recombinant polypeptide, amino acid sequence or nucleic acid sequence, or a fragment or combination of recombinant polypeptides, amino acid sequences or nucleic acid sequences or fragments as described herein.

[0071] In another embodiment, the invention provides a recombinant polypeptide comprising a KLK3 peptide operably linked to an LLO peptide that increases the immunogenicity of the KLK3 peptide.

[0072] As provided herein, the recombinant Listeria strain expressing the LLOKLK3 fusion protects mice against cancer and induces the formation of antigen-specific

-15CTL. Thus, Listeria strains expressing prostate-specific antigens (e.g., prostate-specific antigen / KLK3) are antigenic and effective in vaccination methods. In addition, fusions of LLO and its fragments to prostate-specific antigens (e.g., prostate-specific antigen / KLK3) are antigenic and effective in vaccination methods.

[0073] In addition, as provided herein, Lm-LLO-E7 induces regression of well-established subcutaneous immortalized HPV-16 tumors in C57B1 / 6 mice (Example 1). In addition, as provided herein, Lm-LLO-NP protects mice against RENCA-NP, a renal cell carcinoma (Example 3). In addition, as disclosed herein, fusion of antigens with ActA and PEST-like sequences gives similar results. Hence, non-hemolytic LLO-like, ActA and PEST-like sequences are all effective in increasing the immunogenicity of KLK3, PSCA and FOLH1 peptides.

[0074] In another embodiment, the invention provides a vaccine comprising the recombinant fusion polypeptide of the invention and an adjuvant.

[0075] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention.

[0076] In another embodiment, the invention provides a recombinant vaccine vector encoding a recombinant fusion polypeptide of the invention.

[0077] In another embodiment, the invention provides a nucleotide molecule encoding a recombinant fusion polypeptide of the invention.

[0078] In another embodiment, the invention provides a vaccine comprising the nucleotide molecule of the invention and an adjuvant.

[0079] In another embodiment, the invention provides an immunogenic composition comprising a nucleotide molecule of the invention.

[0080] In another embodiment, the invention provides a recombinant vaccine vector containing the nucleotide molecule of the invention.

[0081] In other embodiments, the adjuvant of the methods and compositions of the invention is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. In another embodiment, the adjuvant is KLH. Recombinant GM-CSF means human protein cultured, in another embodiment, in a yeast vector (S. cerevisiae). GM-CSF stimulates clonal expansion and differentiation of hematopoietic progenitor cells, APCs, dendritic cells and T cells.

[0082] The adjuvant may be a cytokine, growth factor, cell population, QS21, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, BCG, alum, interleukin, unmethylated CpG oligonucleotide, feather glycosides, monophosphorylated lipid A, liposomes, bacterial mitogen or chemokine. In another embodiment, the adjuvant means any other type of adjuvant known in the art. In another form

The vaccine according to the invention contains two of the above adjuvants. In another embodiment, the vaccine contains more than two of the above adjuvants.

[0083] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain of the invention for use in inducing an anti-KLK3 immune response in a subject.

[0084] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain of the invention for use in treating a KLK3-expressing tumor in a subject. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0085] In one embodiment, "treatment" refers to both therapeutic and prophylactic treatment, or preventative measures, the goal being to prevent or reduce a targeted pathological condition or disorder as described herein. Thus, in one embodiment, the treatment may include direct effect or treatment, suppression, inhibition, prevention, decrease in severity, delaying onset, reduction of symptoms associated with the disease, disorder or condition, or combinations thereof. Hence, in one embodiment, "treatment" refers, inter alia, to delaying progression, accelerating remission, inducing remission, increasing remission, accelerating healing, increasing efficacy, or reducing resistance to alternative therapeutics, or a combination thereof. In one embodiment, "preventing" or "obstructing" refers, inter alia, to delaying the onset of symptoms, preventing relapses, reducing the number or frequency of relapse episodes, increasing latency between symptomatic episodes or a combination thereof. In one embodiment, "suppression" or "inhibition" or "protection against" refers, inter alia, to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing symptom latency, reducing symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival or combinations thereof.

[0086] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain of the invention for use in protecting a human subject from a KLK3-expressing tumor. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0087] Methods for assessing the efficacy of prostate cancer vaccines are well known in the art, and are described, for example, in Dzojic H et al. (Adenovirusmediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model. Prostate. 2006 Jun 1; 66 (8): 831-8), Naruishi K et al. (Adenoviral vector-mediated RTVP-1 gene-modified tumor cell-based vaccine suppresses the development of experimental prostate cancer. Cancer Gene Ther. 2006 Jul; 13 (7): 658-63), Sehgal I et al. (Cancer Cell Int. 2006 Aug 23; 6: 21), and Heinrich JE et al. (Vaccination against

-17 Simple cancer using a live tissue factor deficient cell line in Lobund-Wistar rats. Cancer Immunol Immunother 2007; 56 (5): 725-30).

[0088] In another embodiment, the prostate cancer model used to test the methods and compositions of the invention is the mouse PSA-expressing TRAMP-C1 tumor model (TPSA23). In another embodiment, the prostate cancer model is a BMA 178-2 cell model. In another embodiment, the prostate cancer model is a PAIII adenocarcinoma cell model. In another embodiment, the prostate cancer model is the PC-3M model. In another embodiment, the prostate cancer model is any other prior art prostate cancer model.

[0089] In another embodiment, the vaccine is tested in humans, and the efficacy is monitored using methods well known in the art, e.g., direct measurement of CD4 T cell response⁺ and CD8⁺ or measuring disease progression, e.g. by determining the number or size of tumor metastases, or monitoring the symptoms of the disease (cough, chest pain, weight loss, etc.). Methods for assessing the efficacy of a vaccine against prostate cancer in humans are well known in the art, and are described, for example, in Uenaka A et al. (T cell immunomonitoring and tumor responses in patients immunized with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein. Cancer Immun. 2007 Apr 19; 7: 9) and Thomas-Kaskel AK et al. (Vaccination of advanced prostate cancer patients with PSCA and PSA peptide-loaded dendritic cells induces DTH responses that correlate with superior overall survival. Int J Cancer. 2006 Nov 15; 119 (10): 2428-34).

[0090] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention for use in inducing an anti-KLK3 immune response in a subject.

[0091] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention, for use in treating a KLK3-expressing tumor in a subject. In an embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0092] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention for use in protecting a human subject from a KLK3-expressing tumor. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0093] In another embodiment, the invention provides a nucleotide molecule according to the invention for use in inducing an anti-KLK3 immune response in a subject.

[0094] In another embodiment, the invention provides an immunogenic composition comprising a nucleotide molecule of the invention, for use in treating a KLK3-expressing cancer in a subject. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0095] In another embodiment, the invention provides an immunogenic composition comprising a nucleotide molecule of the invention for use in protecting a human subject against a KLK3-expressing cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0096] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain, wherein the strain comprises a recombinant fusion polypeptide of the invention, for use in inducing an anti-KLK3 immune response in a subject.

[0097] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain, wherein the strain comprises a recombinant fusion polypeptide of the invention, for use in treating KLK3-expressing cancer in a subject. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0098] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain, wherein the strain comprises a recombinant fusion polypeptide of the invention, for use in protecting a human subject from KLK3 expressing cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer. In another embodiment, the KLK3-expressing cancer is KLK3-expressing prostate cancer.

[0099] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain of the invention, for use in inhibiting tumor growth - KLK3-expressing prostate cancer in a subject.

[0100] In another embodiment, the invention provides a composition comprising a recombinant Listeria strain of the invention, for use in overcoming immunological tolerance in a subject for cancer - KLK3-expressing prostate cancer. In another embodiment, the invention provides a combination of therapies that in one embodiment include so-called priming [first contact of lymphocytes with a given antigen] by means of Listeria and intensification of the original response [ang. boosting] with a polypeptide.

[0101] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention, for use in

-19 inhibiting tumor growth - KLK3-expressing prostate cancer in a subject.

[0102] In another embodiment, the invention provides an immunogenic composition comprising a recombinant fusion polypeptide of the invention, for use in overcoming immune tolerance in a subject for cancer - KLK3-expressing prostate cancer.

[0103] In another embodiment, the invention provides an immunogenic composition comprising a nucleotide molecule of the invention, for use in inhibiting tumor growth - KLK3-expressing prostate cancer in a subject.

[0104] In another embodiment, the invention provides an immunogenic composition comprising a nucleotide molecule of the invention, for use in overcoming immune tolerance in a subject for cancer - KLK3-expressing prostate cancer.

[0105] "Tolerance" refers, in another embodiment, to the host's lack of reactivity to the antigen. In another embodiment, the term refers to the lack of detectable host reactivity to the antigen. In another embodiment, the term refers to the lack of immunogenicity of the antigen in a given host. In another embodiment, tolerance was measured by inactivity in the CTL assay in vitro. In another embodiment, tolerance was measured by a lack of reactivity in a delayed-type hypersensitivity assay. In another embodiment, tolerance was measured by the lack of reactivity in any other suitable assay known in the art. In another embodiment, tolerance is determined or measured as shown in the examples herein.

[0106] "Overcoming" refers, in another embodiment, to reversing vaccine tolerance. In another embodiment, the term refers to the vaccine conferring a detectable immune response. In another embodiment, the overcoming of immune tolerance is determined or measured as outlined in the examples herein.

[0107] In another embodiment, the invention provides a KLK3-expressing Listeria strain of the invention for use in treating or delaying the progression of benign prostatic hyperplasia (BPH) in a subject.

[0108] In another embodiment, the invention provides a KLK3-expressing Listeria strain of the invention for use in treating or delaying the progression of endothelial prostate (PIN) tumors in a subject.

[0109] In another embodiment, the invention provides a KLK3-containing fusion peptide of the invention for use in treating or delaying BPH progression in a subject.

[0110] In another embodiment, the invention provides a KLK3-containing fusion peptide of the invention for use in treating or delaying PIN progression in a subject.

[0111] In another embodiment, the invention provides a nucleotide molecule encoding a KLK3 fusion protein of the invention, for use in treating or delaying BPH progression in a subject.

[0112] In another embodiment, the invention provides a nucleotide molecule encoding a KLK3 fusion protein of the invention, for use in treating or delaying the progression of endothelial prostate tumors in a subject.

[0113] In another embodiment, the fusion proteins of the invention need not be expressed by LM, but rather can be expressed and isolated from other vectors and cell systems used for protein expression and isolation.

[0114] As provided herein, LLO-E7 fusions show significant therapeutic efficacy. In these experiments, a vaccinia vector was constructed that expresses E7 as a fusion protein with a nonhemolytic truncated form of LLO. Expression of the ILO-E7 fusion product in plaque purified vaccinia was confirmed by Western blotting using an antibody directed against the LLO protein sequence. VacLLO-E7 has been shown to produce CD8 T cells⁺ specific for LLO and E7 as determined using LLO (91-99) and E7 (49-57) epitopes in Balb / c and C57 / BL6 mice, respectively. The results were confirmed in a CTL assay (Example 4).

[0115] Hence, the expression of an antigen, e.g. KLK3, PSCA or FOLH1, as a fusion protein with a nonhemolytic truncated form of LLO-like, ActA or PEST-like sequences in host cell systems in Listeria and host cell systems other than Listeria results in increased antigen immunogenicity. While comparative experiments were performed for vaccinia, a number of other plasmids and expression systems are known that can be used to express these fusion proteins. For example, bacterial vectors useful in the invention include, but are not limited to, Salmonella sp., Shigella sp., BCG, L. monocytogenes and S. gordonii. In addition, the fusion proteins can be delivered using recombinant bacterial vectors modified to avoid fagolysosome fusion and to localize in the cytoplasm of the cell. Viral vectors useful in the invention include, but are not limited to, vaccinia, avipox virus, adenovirus, AAV, vaccinia virus NYVAC, modified vaccinia strain Ankara (MVA), Semliki Forest virus, Venezuelan Equine Encephalitis virus, herpes viruses and retroviruses. Naked DNA vectors can also be used.

[0116] In another embodiment, the KLK3 protein expressed by the target tumor cells has complete homology with the KLK3 peptide (over the entire length of the peptide) expressed by the Listeria vector. In another embodiment, the KLK3 protein is highly homologous (over the entire peptide length) to the KLK3 peptide expressed by the Listeria vector. "Highly homologous" refers, in another embodiment, to homology greater than 90%. In another embodiment, the term refers to homology greater than 92%. In another embodiment, the term refers to homology greater than 93%. In another embodiment, the term refers to homology greater than 94%. In another embodiment, the term refers to homology greater than 95%. In another embodiment, the term refers to homology greater than 96%. In another embodiment, the term refers to homology greater than 97%. In another embodiment, the term refers to homology greater than 98%. In another embodiment, the term refers to homology greater than 99%. In another embodiment, the term refers to 100% homology.

[0117] The KLK3 peptide of the fusion protein of the invention means, in another embodiment, 240-261 amino acids (AA) in length. In another embodiment, the length is 250-261 AA. In another embodiment, the length is about 240 AA. In another embodiment, the length is about 260 AA.

[0118] In another embodiment, the KLK3 fragment consists of about 90% of the KLK3 protein. In another embodiment, the fragment consists of about 95% of the same. In another embodiment, the fragment consists of about 100% thereof.

[0119] In another embodiment, the KLK3 fusion peptide of the invention is an immunogenic peptide. "Immunogenic" refers, in another embodiment, to the ability to induce an immune response when administered to a subject. In another embodiment, the subject means a human subject. In another embodiment, the induced immune response is a T cell response. In another embodiment, the induced immune response is a cytotoxic T lymphocyte (CTL) response. In another embodiment, an induced immune response is detectable. In another embodiment, the induced immune response is detectable by an in vitro assay. In another embodiment, the assay is a cytokine release assay (e.g., fluorescence activated cell sorting; or FACS). In another embodiment, the assay means a chromium release assay or other in vitro cytotoxicity assay.

[0120] In another embodiment, the invention provides a vaccine, immunogenic composition or vector comprising the recombinant Listeria strain of the invention for use in reducing the size of a KLK3-expressing tumor. In another embodiment, the tumor cell expresses KLK3.

[0121] In another embodiment, the invention provides an effective amount of a vaccine comprising any of the following: (a) a recombinant Listeria strain comprising an N-terminal fragment of LLO protein conjugated to KLK3 peptide; or (b) a recombinant nucleotide encoding a recombinant polypeptide for use in inhibiting the formation of a KLK3-expressing tumor.

[0122] In another embodiment, the invention provides a vaccine, immunogenic composition or vector comprising the recombinant fusion polypeptide of the invention for use in reducing the size of a KLK3-expressing tumor. In another embodiment, the tumor cell expresses KLK3.

[0123] In another embodiment, the invention provides an effective amount of a vaccine comprising any of the following: (a) a recombinant polypeptide comprising an N-terminal fragment of LLO protein conjugated to KLK3 peptide; or (b) a recombinant nucleotide encoding a recombinant polypeptide for use in inhibiting the formation of a KLK3-expressing tumor.

[0124] In another embodiment, the invention provides a vaccine, immunogenic composition or vector comprising the recombinant nucleotide molecule of

Of the invention for use in reducing the size of a KLK3-expressing tumor. In another embodiment, the tumor cell expresses KLK3.

[0125] In another embodiment, the invention provides an effective amount of a vaccine comprising any of the following: (a) a recombinant nucleotide molecule comprising the N-terminal fragment of the LLO protein coupled to the KLK3 peptide; or (b) a recombinant nucleotide encoding a recombinant polypeptide for use in inhibiting the formation of a KLK3-expressing tumor.

[0126] In the invention, the recombinant fusion peptide for medical uses and compositions of the invention is an LLO-KLK3 fusion peptide comprising the sequence given in SEQ ID No: 54. In another embodiment, the fusion peptide has the sequence given in SEQ ID No: 54. In another In one embodiment, the fusion peptide is homologous to the sequence given in SEQ ID No: 54, and has a percentage of identity with SEQ ID No: 54 greater than 99%.

[0127] The sequence of the LLO protein used in the construction of the vaccines of the invention means, in another embodiment, a fragment of SEQ ID NO: 17 (GenBank accession number P13128; the nucleic acid sequence is given under GenBank accession number X15127). The first 25 amino acids of the pro-protein corresponding to this sequence are the signal sequence and are cleaved from LLO after secretion by the bacterium. Hence, in this embodiment, the active, full-length LLO protein is 504 residues in length.

[0128] In another embodiment, the "LLO peptide" and "LLO fragment" refer to the N-terminal fragment of the LLO protein. In another embodiment, the terms refer to full-length but non-hemolytic LLO protein. In another embodiment, the terms refer to a nonhemolytic protein containing a point mutation in cysteine ​​484 from ID No: 17 or the corresponding residue in the homologous LLO protein.

[0129] In another embodiment, the N-terminal fragment of LLO protein used in the compositions and methods of the invention has the sequence SEQ ID NO: 18.

[0130] As disclosed herein, recombinant Listeria strains expressing the fusion of PEST-like sequences with an antigen induce anti-tumor immunity (Example 5) and generate antigen-specific tumor infiltrating T cells (Example 6).

[0131] In another embodiment of the invention, "nucleic acids" or "nucleotide" refers to a string of at least two base-sugar-phosphate combinations. The term includes, in one embodiment, DNA and RNA. "Nucleotides" refers, in one embodiment, to monomeric units of nucleic acid polymers. RNA may exist, in one embodiment, in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (matrix RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al., Genes & Devel 16: 2491-96; and in the literature cited therein). DNA may be in the form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA can be single, double, triple or quadruple strands.

The term also includes, in another embodiment, artificial nucleic acids, which may contain other types of skeletons, but the same bases. In one embodiment, the artificial nucleic acid is PNA (peptide nucleic acid). PNA contains peptide backbones and nucleotide bases and are capable of binding, in one embodiment, both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane-modified. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester linkages with a phosphorothioate linkage. In another embodiment, the artificial nucleic acid comprises any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothioate nucleic acids and PNA is known to those skilled in the art, and is described in, for example, Neilsen PE, Curr Opin Struct Biol 9: 353-57; and Raz NK et al. Biochem Biophys Res Commun. 297: 1075-1084. The production and use of nucleic acids is known to those of skill in the art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. And Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and GC Fareed.

[0132] Also described herein is a kit containing the reagent used in carrying out the invention. In another embodiment, the invention provides a kit comprising a composition, tool or instrument of the invention.

[0133] In another embodiment, the LLO fragment is attached to KLK3 peptide by chemical coupling. In another embodiment, paraformaldehyde is used for coupling. In another embodiment, maleimide is used for coupling. In another embodiment, the coupling is carried out using any suitable method known in the art.

[0134] In another embodiment, the KLK3-expressing tumor to which the compositions of the invention are directed is KLK3-expressing prostate cancer. In another embodiment, the KLK3 expressing tumor is KLK3 expressing prostate cancer.

[0135] As disclosed herein, increased cellular immunity has been demonstrated in antigen-containing and truncated LLO fusion proteins containing a PEST-like amino acid sequence, SEQ ID NO: 1. Al1010 used in some examples was 416 amino acids in length (after cleavage of the signal peptide) as 88 residues from the carboxy terminus, which includes the activation domain containing 484 cysteine, were cleaved. However, it is evident from the disclosure that other ΙΙΟΙΙΟs without the activation domain and in particular cysteine ​​484 are effective in the methods of the invention.

[0136] As provided herein, the fusion of antigen with the non-haemolytic truncated form of listeriolysin O (llO) increases immunogenicity. The 1M vector that expresses and secretes the fusion product of the human papillomavirus (HPV) 16 E7 and 10O was a more potent cancer immunotherapeutic for HPV-immortalized tumors than 1M only secreting E7 protein. In addition, the recombinant vaccinia virus that carries the gene for the 10-E7 fusion protein is a stronger cancer immunotherapist for HPV-immortalized tumors than the isogenic strain of vaccinia that carries the gene for E7 protein alone. For

Compared, the short Lm-AZ / -E7 fusion protein containing the E7 antigen coupled to the promoter, signal sequence and first 7 AA LLO residues was an ineffective anti-tumor immunotherapeutic. This short fusion protein is terminated immediately before and does not contain a PEST-like sequence.

[0137] "Fusion protein" refers, in another embodiment, to a protein comprising 2 or more proteins linked together by peptide bonds or other chemical bonds. In another embodiment, the proteins are linked together directly through a peptide bond or other chemical bond. In another embodiment, the proteins are linked together with one or more amino acids (e.g., "distance") between two or more proteins.

[0138] Fusion proteins containing KLK3 peptide are, in another embodiment, obtained in any suitable manner. In another embodiment, the fusion protein is obtained by cloning and restriction cleavage of the respective sequences or direct chemical synthesis, as discussed below. In another embodiment, the sequences are cloned and the respective sequences are cleaved using the appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to give the desired DNA sequence. In another embodiment, the DNA encoding the KLK3 peptide is produced using DNA amplification methods, for example by polymerase chain reaction (PCR). First, segments of native DNA on either side of the new end are amplified separately. The 5 'end of one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. As the 5 'end of the first fragment is complementary to the 3' end of the second fragment, two fragments (after partial purification, e.g. on LMP agarose) can be used as the overlapping template in the third PCR reaction. The amplified sequence will contain codons, a segment on the carboxyl side of the opening site (currently forming the amino sequence), a linker, and a sequence on the amino side of the opening site (currently forming the carboxyl sequence). The gene encoding KLK3 peptide is then ligated into the plasmid.

[0139] In another embodiment, the KLK3 peptide is coupled to the truncated LLO protein by any of the well-known solutions of the skilled person. In another embodiment, the KLK3 peptide is conjugated, directly or via a linker (spacer), to the protein. In another embodiment, wherein both KLK3 peptide and LLO protein are polypeptides, the chimeric molecule is expressed recombinantly as a single chain fusion protein.

[0140] In another embodiment, the peptides and proteins of the invention are readily obtained by standard, well-established solid phase peptide synthesis (SPPS) as described in Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill .; and as described in Bodanszky and Bodanszky (The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York). First, a properly protected amino acid residue is attached via its carboxyl group to a derivatized, insoluble polymeric substrate, such as cross-linked polystyrene resin or

-25poliamidowa. "Properly protected" refers to the presence of protecting groups on both alpha-amino acid groups and on the functional groups of any side chains. The side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used during the synthesis, and are removable under conditions that will not affect the final peptide product. Stepwise oligopeptide synthesis is carried out by removing the N-protecting group from the initial amino acid, and coupling it to the carboxy terminus of the next amino acid in the sequence of the desired peptide. This amino acid is also properly protected. The carboxy of the further amino acid can be activated to react with the N-terminus of the substrate-associated amino acid by forming a reactive group, such as the formation of a carbodiimide, a symmetrical acid anhydride or an "active ester" group such as hydroxybenzotriazole or pentafluorophenyl ester.

[0141] Examples of solid phase peptide synthesis methods include the BOC method that uses tert-butyloxycarbonyl as a protecting group for alpha-amino, and the FMOC method that uses 9-fluorenylmethyloxycarbonyl to protect alpha-amino acid residues, both of which are good known to experts.

[0142] Incorporation of N- and / or C-blocking groups can also be achieved using conventional protocols for solid phase peptide synthesis methods. For incorporation of C-terminal blocking groups, for example, the synthesis of the desired peptide is typically carried out using, as a solid phase, a resin medium that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C terminus carries a primary amine blocking group, for example, synthesis is carried out using p-methylbenzhydrylamine (MBHA) resin so that when peptide synthesis is completed, hydrofluoric acid treatment releases the desired C-terminal amidated peptide. Similarly, incorporation of the N-methylamino blocking group at the C-terminus is achieved using N-methylaminoethyl derivatized DVB, a resin that releases a N-methylamidated C-bearing peptide after HF treatment. C-terminus blocking can also be achieved by esterification using conventional procedures . This includes the use of a resin / blocking group combination that allows the peptide side chain to be released from the resin to allow subsequent reaction with the desired alcohol to form an ester function. The FMOC protecting group, in combination with a DVB resin derivatized with methoxyalkoxybenzyl alcohol or an equivalent linker, can be used for this purpose, with substrate cleavage by TFA in dichloromethane. Esterification of an appropriately activated carboxyl function e.g. DCC can then proceed by the addition of the desired alcohol followed by deprotection and isolation of the esterified peptide product.

[0143] Incorporation of N-terminal blocking groups can be achieved while the synthesized peptide is still attached to the resin, for example by treatment with the appropriate anhydride and nitrile. To incorporate the N-terminal acetyl blocking group, for example, the resin-coupled peptide can be treated with 20% anhydride

-26 acetic acid in acetonitrile. The N-blocked peptide product can then be cleaved from the resin, deprotected and then isolated.

[0144] In another embodiment, to ensure that the peptide obtained by chemical or biological synthesis techniques is the desired peptide, peptide composition analysis is performed. In another embodiment, amino acid composition analysis is performed using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or in addition, the amino acid composition of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequencers that sequentially degrade the peptide and identify the amino acids in turn can also be used to definitively determine the peptide sequence.

[0145] In another embodiment, before using it, the peptide is purified to remove impurities. In this regard, it will be recognized that the peptide will be purified to meet the standards provided by the relevant regulatory agencies and guidelines. Any of a number of conventional purification procedures can be used to achieve the required level of purity, including, for example, high performance reverse phase liquid chromatography (HPLC) using an alkylated silica column, such as C4-, C8- or C18-silica. Generally, a mobile phase gradient with increasing organic content is used to achieve purification, for example, acetonitrile in aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion exchange chromatography can also be used to separate peptides based on their charge.

[0146] Solid phase synthesis is used in which the C-terminal AA of the sequence is attached to an insoluble support, followed by subsequent addition of the remaining amino acids in the sequence, in another embodiment, for the chemical synthesis of the polypeptides of the invention. Solid phase synthesis techniques are described in Barany and Merrifield in SolidPhase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al. J. Am. Chem. Soc., 85: 21492156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd edition. Pierce Chem. Co., Rockford, Ill. (1984).

[0147] In another embodiment, the peptides of the invention can incorporate AA residues that are modified without affecting activity. For example, the ends can be derivatized to contain blocking groups, i.e. chemical substituents suitable for protecting and / or stabilizing the N- and C-termini against "undesirable degradation" - a term intended to include any type of enzymatic, chemical or biochemical degradation of a compound at its ends that is likely to affect the functions of the compound, i.e. sequential degradation of the compound at its ends.

[0148] In another embodiment, the blocking groups include protecting groups conventionally used in the field of peptide chemistry that will not adversely affect peptide activities in vivo. For example, the corresponding N-terminal groups

Blocking agents can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as their substituted forms such as acetamidomethyl group (Acm). The amino acid desamino analogs are also useful N-terminal blocking groups, and can be coupled to the N-terminus of the peptide or used instead of the N-terminal residue. Suitable C-terminal blocking groups in which the C-terminal carboxy group is incorporated or not incorporated include esters, ketones or amides. Esters or ketones forming alkyl groups, certain lower alkyl groups such as methyl, ethyl and propyl, and amide forming amines such as primary amines (- NH2), and mono- and di-alkyl amino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. De-carboxylated amino acid analogs such as agmatine are also useful C-terminal blocking groups and can be coupled to the C-terminal residue of the peptide or used instead. In addition, it will be appreciated that the free amino and carboxy groups at the ends can be completely removed from the peptide, resulting in de-amino and de-carboxylated forms, without affecting peptide activity.

[0149] In another embodiment, other modifications are incorporated without adversely affecting the activity. In another embodiment, such modifications include, but are not limited to, the substitution of one or more amino acids in their natural lysomeric form for amino acids in their D-isomeric form. Hence, the peptide may contain one or more D-amino acid residues or may contain amino acids that all exist in the D form. Retro-inverse peptide forms of the invention are also contemplated, for example inverted peptides in which all amino acids are substituted with D-amino acid forms.

[0150] In another embodiment, the acid addition salts of the peptides of the invention are used as their functional equivalents. In another embodiment, the peptide according to the invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric acid, etc., or an organic acid such as acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, amber, maleic, fumaric, tartaric, lemon, benzoic, cinnamon, almond, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, etc., to provide a water-soluble peptide salt is suitable for use in the invention.

[0151] In another embodiment, the modifications (which do not usually change the primary sequence) include chemical derivatization in vivo, or in vitro of the polypeptides, e.g. acetylation or carboxylation. Also included are glycosylation modifications, e.g., those obtained by modifying glycosylation patterns of a polypeptide during synthesis and processing, or at subsequent processing stages; e.g. by exposing the polypeptide to enzymes that affect glycosylation, e.g. mammalian glycosylating or deglycosylating enzymes. Also included

- sequences that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine or phosphotreonin.

[0152] In another embodiment, the polypeptides are modified using conventional molecular biology techniques to improve their resistance to proteolytic degradation or to optimize solubility properties, or to make them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring 1-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary methods detailed herein.

[0153] In another embodiment, the chimeric fusion proteins of the invention are synthesized using recombinant DNA methodology. This generally involves the creation of a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette, such as the plasmid of the invention, under the control of a particular promoter / regulatory element, and protein expression. The DNA encoding the fusion protein of the invention is obtained, in another embodiment, by any suitable method, including, for example, cloning and restriction cleavage of the respective sequences or direct chemical synthesis by methods such as the phosphotriester method from Narang et al. (1979, Meth. Enzymol. 68: 90-99); the phosphodiester method from Brown et al. (1979, Meth. Enzymol 68: 109151); diethylamidophosphite method from Beaucage et al. (1981, Tetra. lett., 22: 1859-1862); and a method on a solid support according to US Patent No. 4,458,066.

[0154] Chemical synthesis produces a single-stranded oligonucleotide. It is transformed, in another embodiment, into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with DNA polymerase using a single strand as a template. The skilled person will recognize that - while chemical synthesis of DNA is limited to a sequence of about 100 bases, longer sequences can be obtained by ligation of shorter sequences.

[0155] In another embodiment, an "isolated nucleic acid" includes RNA or DNA sequences encoding a fusion protein of the invention, and any modified forms thereof, including chemical modifications of DNA or RNA that make the nucleotide sequence more stable when it is cell free or when associated with a cell. Chemical modification of nucleotides can also be used to increase the efficacy with which a nucleotide sequence is captured by a cell or the efficacy with which it is expressed in a cell. Such modifications are described in detail elsewhere in the description. Any and all combinations of nucleotide sequence modifications are contemplated in the present invention.

[0156] In another embodiment, the invention provides an isolated nucleic acid encoding a KlK3 peptide operably linked to a nonhemolytic IL10 sequence, wherein the isolated nucleic acid further comprises a promoter / regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid . Hence, the invention includes expression vectors and methods for introducing exogenous DNA into cells, with the simultaneous expression of exogenous DNA in cells,

Such as those described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0157] In another embodiment, the nucleotide of the invention is operably linked to a promoter / regulatory sequence that directs expression of the encoded peptide in the Listeria strain. Promoter / regulatory sequences useful for directing constitutive gene expression are well known in the art and include, but are not limited to, for example, the PhlyA, PActA and p60 promoters from Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter and the B. thuringiensis phaZ promoter. It will therefore be appreciated that the invention includes the use of any promoters / regulatory sequences that are capable of directing the expression of the desired protein operably linked thereto.

[0158] Expression of the KLK3 peptide operably linked to a nonhemolytic LLO sequence using a vector allows the isolation of large amounts of recombinantly produced protein. It is within the skill of the skilled person to select specific promoters / regulatory sequences and to functionally link these promoters / regulatory sequences to the DNA sequence encoding the desired polypeptide. This technology is well known in the art and is described for example in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0159] In another embodiment, the invention provides a vector comprising an isolated nucleic acid encoding a KLK3 peptide operably linked to a nonhemolytic LLO sequence. Incorporation of the desired nucleic acid into the vector and selection of vectors are well known in the art as described in, for example, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0160] In another embodiment, the invention provides cells, viruses, proviruses, etc., containing such vectors. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See. for example, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0161] In another embodiment, the nucleic acids encoding the KLK3 peptide operably linked to a nonhemolytic LLO sequence are cloned into a plasmid vector. In another embodiment, the recombinant Listeria strain is transformed with a plasmid vector.

[0162] When disposing of the invention, it is clearly apparent to those skilled in the art that other nucleic acids encoding KLK3 peptide operably linked to a nonhemolytic LLO sequence can be obtained by following the procedures described in the description in the detailed experimental section for generating other fusion proteins as disclosed

In the description (e.g., directed mutagenesis, reading frame shift mutations, etc.), and prior art procedures.

[0163] Methods for generating derivatives or forms of variants of fusion proteins are well known in the art, and include, inter alia, the use of recombinant DNA methodology well known in the art, such as, for example, that described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York) and elsewhere in the description.

[0164] In another embodiment, the invention provides a nucleic acid encoding KLK3 peptide operably linked to a nonhemolytic LLO sequence, wherein the nucleic acid encoding the polypeptide tag is covalently attached thereto. That is, the invention includes a chimeric nucleic acid, wherein the nucleic acid sequence encoding the polypeptide tag is covalently linked to the nucleic acid encoding the protein containing KLK3 peptide. Such polypeptide markers are well known in the art and include, for example, green fluorescent protein (GFP), myc, pyruvate myc-kinase (myc-PK), His6, maltose binding protein (MBP), influenza hemagglutinin tag polypeptide. polypeptide Flag tag (FLAG) and glutathione-S-transferase polypeptide tag (GST). However, the invention should under no circumstances be interpreted as being limited to the nucleic acids encoding the polypeptide markers listed above. Rather, any nucleic acid sequence encoding a polypeptide that can function using a solution that is substantially similar to those polypeptide tags should be interpreted as encompassed by the present invention.

[0165] In another embodiment, the invention provides a KLK3 fusion peptide with increased immunogenicity. That is, as the data disclosed herein show, the KLK3 peptide conjugated to the truncated nonhemolytic LLO protein, after administration to the animal, results in the clearance of existing tumors and the induction of antigen-specific cytotoxic lymphocytes capable of infiltrating the tumor or infected cells. After reading the disclosure and the medical uses and compositions disclosed herein, one of ordinary skill in the art will readily understand that the invention is suitable for the treatment and / or prevention of many diseases.

[0166] In another embodiment, a commercially available plasmid is used in the invention. Such plasmids are available from a number of sources, for example, Invitrogen (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), Clontech (Palo Alto, Calif.), Or can be constructed using methods well known in the art. A commercially available plasmid such as pET (Gibbstown, NJ) is a prokaryotic expression vector with a prokaryotic replication origin and promoter / regulatory elements to facilitate expression in a prokaryotic organism.

[0167] The invention further includes transforming, such as a Listeria strain, a plasmid containing (a) KLK3 peptide; and (b) isolated nucleic acid encoding the truncated LLO protein. In another embodiment, if the LM vaccine strain contains a deletion in the prfA or actA gene, the plasmid contains the prfA or actA gene for mutation complementation, thereby restoring the function of the L. monocytogenes vaccine to the strain. As described elsewhere

As described herein, methods for transforming bacteria are well known in the art, and include methods based on calcium chloride, competent cells, electroporation methods, bacteriophage-mediated transduction, chemical and physical transformation techniques (de Boer et al., 1989, Cell 56: 641 -649; Miller et al., 1995, FASEB J., 9: 190-199; Sambrook et al. 1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., Eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC; Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY).

[0168] The plasmid of the invention comprises, in another embodiment, a promoter / regulatory sequence operably linked to the gene encoding the fusion protein.

[0169] Plasmids and other expression vectors useful in the invention are described elsewhere in the description, and may contain functions such as promoter / regulatory sequence, origin of replication for Gram-negative and / or Gram-positive bacteria, and isolated nucleic acid encoding fusion protein. In addition, an isolated nucleic acid encoding a fusion protein will have its own promoter suitable for directing the expression of such an isolated nucleic acid. Promoters useful for directing expression in a bacterial system are well known in the art, and include lambda bacteriophage, the bla beta-lactamase gene promoter from pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene from pBR325. Further examples of prokaryotic promoters include the main right and left promoters of bacteriophage lambda (P1 and PR), promoters trp, recA, lacZ, lacd and gal from E. coli, alpha-amylase promoters (Ulmanen et al, 1985. J. Bacteriol. 162: 176-182) and S28-specific promoters from B. subtilis (Gilman et al, 1984 Gene 32: 11-20), Bacillus bacteriophage promoters (Gryczan, 1982, in: Molecular Biology of the Bacilli, Academic Press, Inc. , New York), and Streptomyces promoters (Ward et al, 1986, Mol. Gen. Genet. 203: 468-478). Additional prokaryotic promoters contemplated by the invention are discussed in, for example, Glick (1987, J. Ind. Microbiol. 1: 277-282); Cenatiempo, (1986, Biochimie, 68: 505-516); and Gottesman, (1984, Ann. Rev. Genet. 18: 415-442). Further examples of promoter / regulatory elements contemplated by the invention include, but are not limited to, the listeria prfA promoter (GenBank accession no. Y07639), the hly listeria promoter (GenBank accession no. X15127) and the p60 listeria promoter (GenBank accession no. AY126342), or fragments thereof.

[0170] In another embodiment, the Listeria strain of the invention comprises an integrated gene encoding a peptide that comprises a KlK3 fusion peptide.

[0171] In another embodiment, the Listeria strain of the invention is created using a site-specific integration vector. In another embodiment, the Listeria strain containing the integrated gene is created using homologous recombination. In another embodiment, the Listeria strain containing the integrated gene is created using any other method known in the art to integrate the gene into the Listeria chromosome.

[0172] In another embodiment, the integration vector comprises a PSA attPP 'site. In another embodiment, the integration vector comprises a gene coding for PSA integration. In another form

32 the implementation of the integration vector contains an U153 attPP 'site. In another embodiment, the integration vector comprises a gene encoding the U153 integrase. In another embodiment, the integration vector comprises an A118 attPP 'site. In another embodiment, the integration vector comprises a gene encoding the A118 integrase. In another embodiment, the integration vector comprises any other attPP 'site known in the art. In another embodiment, the integration vector comprises any other phage integration known in the art.

[0173] In another embodiment, the Listeria strain of the methods and compositions of the invention comprises a mutation or auxotrophy in the metabolic gene. In another embodiment, the Listeria strain of the methods and compositions of the invention further comprises a mutation in the endogenous ActA gene. In another embodiment, the plasmid bearing the KLK3 fusion peptide comprises a metabolic gene that complements the mutation or auxotrophy. In another embodiment, the integration vector encoding the KLK3 fusion peptide or construct used for integration into the Listeria chromosome contains a gene that complements the mutation or auxotrophy. In another embodiment, the metabolic gene is used for selection instead of the antibiotic resistance gene. In another embodiment, the metabolic gene is used for selection in addition to the antibiotic resistance gene.

[0174] In another embodiment, the metabolic gene is a gene encoding an amino acid metabolism enzyme. In another embodiment, the metabolic enzyme is alanine racemase (dal) enzyme. In another embodiment, the metabolic enzyme is D-amino acid transferase enzyme (dat).

[0175] In another embodiment, the metabolic enzyme metabolizes the amino acid (AA), which is used in the process of bacterial growth. In another embodiment, the AA product is used in the replication process. In another embodiment, the AA product is used in cell wall synthesis. In another embodiment, the metabolic enzyme catalyzes the formation of an amino acid used in cell wall synthesis. In another embodiment, the metabolic enzyme is an amino acid metabolism enzyme. In another embodiment, the AA product is used in protein synthesis. In another embodiment, the AA product is used in fatty acid metabolism. In another embodiment, the AA product is used in any other growth or replication process known in the art.

[0176] In another embodiment, the metabolic enzyme catalyzes the formation of AA used in cell wall synthesis. In another embodiment, the metabolic enzyme catalyzes the synthesis of AA used in cell wall synthesis. In another embodiment, the metabolic enzyme is involved in the synthesis of AA used in cell wall synthesis. In another embodiment, AA is used in cell wall biogenesis.

[0177] In another embodiment, the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.

[0178] In another embodiment, the metabolic enzyme is encoded by the alanine racemase gene (dal). The synthesis of D-glutamic acid is partly controlled by the gene

-33dal, which is involved in the conversion of D-glu + pyr to alpha-ketoglutarate + D-ala, and the reverse reaction.

[0179] In another embodiment, the protein of the methods and compositions of the invention has the sequence SEQ ID No: 56 (GenBank accession number: AF038438). In another embodiment, the dal protein is homologous to SEQ ID No: 56. In another embodiment, the dal protein is a variant of SEQ ID No: 56. In another embodiment, the dal protein is an isoform of SEQ ID No: 56. In another embodiment, the dal protein is fragment of SEQ ID No: 56. In another embodiment, the dal protein is a fragment of the homologue of SEQ ID No: 56. In another embodiment, the protein is a fragment of a variant of SEQ ID No: 56. In another embodiment, the dal protein is a fragment of the isoform of SEQ ID No: 56.

[0180] In another embodiment, the dal protein is any other Listeria dal protein known in the art. In another embodiment, the dal protein is any Bacillus subtilis dal protein known in the art. In another embodiment, the dal protein is any other gram-positive dal protein known in the art. In another embodiment, the dal protein is any other dal protein known in the art.

[0181] The date protein of the methods and compositions of the invention is encoded, in another embodiment, by SEQ ID No: 57 (GenBank accession number: AF038439). In another embodiment, the dat protein is homologous to SEQ ID No: 57. In another embodiment, the dat protein is a variant of SEQ ID No: 57. In another embodiment, the dat protein is an isoform of SEQ ID No: 57. In another embodiment, the dat protein means a fragment of SEQ ID No: 57. In another embodiment, the dat protein is a homolog fragment from SEQ ID No: 57. In another embodiment, the dat protein is a fragment of a variant from SEQ ID No: 57. In another embodiment, the protein is a fragment of an isoform from SEQ ID No: 57.

[0182] In another embodiment, the protein dat is any other Listeria date protein known in the art. In another embodiment, the dat protein is any other gram-positive dat protein known in the art. In another embodiment, the dat protein is any other date protein known in the art.

[0183] In another embodiment, the metabolic enzyme is a Dglutamic acid synthesis gene. In another embodiment, the metabolic enzyme is encoded by dga. In another embodiment, the metabolic enzyme is encoded by the alr (alanine racemase) gene. In another embodiment, the metabolic enzyme is any other prior art enzyme that is involved in the synthesis of alanine.

[0184] In another embodiment, the metabolic enzyme is encoded by serC, phosphoserine transaminase. In another embodiment, the metabolic enzyme is encoded by asd (aspartate semialdehyde beta-dehydrogenase) involved in the synthesis of the cell wall component diaminopimelic acid. In another embodiment, the metabolic enzyme is encoded by gsaB-glutamate-1-semialdehyde transaminase, which catalyzes the formation of 5-amionolevulinate from (S) -4-amino-5-oxopentanoate. In another embodiment, the metabolic enzyme is encoded by HemL, which catalyzes the formation of 5-amionolevulinate from (S) -4-amino-5-oxopentanoate. In the other

In embodiments, the metabolic enzyme is encoded by aspB, an aspartate transaminase that catalyzes the formation of oxaloacetate and L-glutamate from L-aspartate and 2-oxoglutarate. In another embodiment, the metabolic enzyme is encoded by argF1, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroE, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroB, involved in 3-hydroxyquinate biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroD, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroC, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisB, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisD, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisG, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by metX, involved in methionine biosynthesis. In another embodiment, the metabolic enzyme is encoded by proB, involved in proline biosynthesis. In another embodiment, the metabolic enzyme is encoded by argR, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by argJ, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by thiI, involved in thiamine biosynthesis. In another embodiment, the metabolic enzyme is encoded by LMOf2365_1652, involved in tryptophan biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroA, involved in tryptophan biosynthesis. In another embodiment, the metabolic enzyme is encoded by ilvD, involved in valine and isoleucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by ilvC, involved in valine and isoleucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by leuA, involved in leucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by dapF, involved in lysine biosynthesis. In another embodiment, the metabolic enzyme is encoded by thrB, involved in threonine biosynthesis (all with GenBank accession number

NC_002973).

[0185] In another embodiment, the metabolic enzyme is tRNA synthetase. In another embodiment, the metabolic enzyme is encoded by the trpS gene encoding tryptophanyl-tRNA synthetase. In another embodiment, the metabolic enzyme is any other tRNA synthetase known in the art.

[0186] In another embodiment, the host bacterial strain is A (trpS aroA), and both markers are included in the integration vector.

[0187] In another embodiment, the metabolic enzyme is encoded by murE, involved in the synthesis of diaminopimelic acid (GenBank accession number:

NC_003485).

[0188] In another embodiment, the metabolic enzyme is encoded by LMOf2365_2494, involved in biosynthesis of chichoic acid.

[0189] In another embodiment, the metabolic enzyme is encoded by WecE (lipopolysaccharide biosynthesis - rffA protein; GenBank accession number: AE014075.1). In another embodiment, the metabolic enzyme is encoded by amiA, Nacetylmuramoyl-L-alanine amidase. In another embodiment, the metabolic enzyme is aspartate aminotransferase. In another embodiment, the metabolic enzyme is histidine-phosphate aminotransferase (GenBank accession number NP_466347). In another embodiment, the metabolic enzyme is glycosylation protein of this cell wall glycosylation - GtcA.

[0190] In another embodiment, the metabolic enzyme is a synthetic enzyme for a peptidoglycan component or precursor. In another embodiment, the component is UDPN-acetylmuramyl-pentapeptide. In another embodiment, the component is UDP-N-N-ethylglucosamine. In another embodiment, the component is MurNAc- (pentapeptide) pyrophosphoryl-undecaprenol. In another embodiment, the component is GlcNAc-A- (1,4) MurNAc- (pentapeptide) -pyrophosphoryl-undecaprenol. In another embodiment, the component is any other peptidoglycan component or precursor known in the art.

[0191] In another embodiment, the metabolic enzyme is encoded by murG. In another embodiment, the metabolic enzyme is encoded by murD. In another embodiment, the metabolic enzyme is encoded by murA-1. In another embodiment, the metabolic enzyme is encoded by murA-2 (all given under GenBank accession number NC_002973). In another embodiment, the metabolic enzyme is any other synthetic enzyme for a peptidoglycan component or precursor.

[0192] In another embodiment, the metabolic enzyme is trans-glycoslase. In another embodiment, the metabolic enzyme is trans-peptidase. In another embodiment, the metabolic enzyme is carboxypeptidase. In another embodiment, the metabolic enzyme is any other class of metabolic enzymes known in the art.

[0193] In another embodiment, the metabolic enzyme is any other Listeria monocytogenes metabolic enzyme known in the art.

[0194] In another embodiment, the metabolic enzyme is any other Listeria metabolic enzyme known in the art.

[0195] In another embodiment, the metabolic enzyme is any other gram-positive metabolic enzyme known in the art.

[0196] In another embodiment, the metabolic enzyme is any other metabolic enzyme known in the art.

[0197] In another embodiment, the integration vector is any other site-specific integration vector known in the art that is capable of infecting

Listeria.

[0198] In another embodiment, the invention provides compositions for increasing the immunogenicity of KLK3 antigen by fusion of the antigen to non-haemolytic truncated

36 LLO ("ALLO") characters, such as SEQ ID NO: 1 for LLO. The invention further provides methods and compositions for increasing the immunogenicity of KLK3 antigen by coupling the antigen to a truncated LLO protein. As demonstrated by the data disclosed herein, the antigen coupled to the ActA protein elicits an immune response that removes existing tumors and results in the induction of antigen-specific cytotoxic lymphocytes.

[0199] In another embodiment, the fusion proteins of the invention are produced recombinantly by transcription and translation, in a bacterium, plasmid or nucleotide molecule that encodes both KLK3 peptide and LLO peptide. In another embodiment, the plasmid or nucleotide is transcribed and / or translated in vitro. In another embodiment, the antigen is chemically coupled to a truncated LLO containing the PEST-like AA sequence from L. monocytogenes or PEST-like AA sequence obtained from another prokaryotic organism. "Antigen" refers, in another embodiment, to the native KLK3 gene product or abbreviated versions of those that contain identified T cell epitopes. In another embodiment, these fusion proteins are then incorporated into vaccines, for administration to a subject, to elicit an increased immune response against the protein antigen. fusion. In other embodiments, the fusion proteins of the invention are provided as DNA vaccines, RNA vaccines, or replicating RNA vaccines. As will be apparent to those skilled in the art from the disclosure, vaccines comprising the fusion proteins of the invention are particularly useful in preventing and treating infectious and cancerous diseases.

[0200] The invention further comprises a composition comprising a vaccine of the invention suitable for administration to an animal or human. The composition includes, but is not limited to, a pharmaceutically acceptable carrier. In another embodiment, the composition comprises a Listeria vaccine strain comprising the truncated LLO protein, or a fragment thereof, conjugated to KLK3 peptide, and a pharmaceutically acceptable carrier.

[0201] To the extent that the following Examples do not relate to the fusion of KLK3 peptide and LLO peptide, these are comparative examples.

A DETAILED EXPERIMENTAL PART

EXAMPLE 1: LLO-ANTIGENE MERGERS INDUCT RESISTANCE

antineoplastic

EXPERIMENTAL MATERIALS AND METHODS (EXAMPLES 1-2)

Cell lines [0202] C57BL / 6 syngeneic TC-1 tumor was HPV-16 E6 and E7 immortalized and transformed with c-Ha-ras oncogene. TC-1 expresses low concentrations of E6 and E7 and is highly carcinogenic. TC-1 was grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 100 μΜ non-essential amino acids, 1 mM sodium pyruvate, 50 micromoles (mcM) 2-ME , 400 microgram (mcg) / ml G418, and 10% National Collection Type Culture-109 medium at 37 ° C with 10% CO2. C3 is an embryonic cell

-37 mice from C57Bl / 6 mice immortalized with the complete HPV 16 genome and transformed with pEJ-ras. El-4 / E7 is El-4 thymoma retroviral transduced E7.

Strains and propagation of L. monocytogenes [0203] The Listeria strains used were lm-lO-E7 (hly-E7 fusion gene in the episomal expression system; Fig. 1A), lm-E7 (single copy of the E7 gene cassette integrated into the Listeria genome), lm-10-NP ("DP-l2028"; hly-NP fusion gene in an episomal expression system) and lm-Gag ("ZY-18"; single copy of the HIV-1 Gag gene cassette integrated into the chromosome).

[0204] For pGG-55 generation, plasmid 10O-E7, E7 was PCR amplified using the 5'-GGCTCGAGC ATGGAGAT AC ACC-3 'primers (SEQ ID No: 8; XhoI site underlined) and 5'-GGGGACTAGTTTATGGTTTTGGTAC' (SEQ ID No: 9; SpeI site underlined) and ligated into pCR2.1 (Invitrogen, San Diego, CA). E7 was excised from pCR2.1 by XhoI / SpeI digestion and ligated into pDP-2028 (Ikonomidis G et al. Delivery of a viral antigen to the class I processing and presentation pathway by listeria monocytogenes. J Exp Med. 1994 Dec 1; 180 (6): 2209-18). The hly-E7 fusion gene and pluripotent transcription factor prfA were amplified and subcloned into pAM401, a multicopy shuttle plasmid (Wirth R et al., J Bacteriol, 165: 831, 1986), generating pGG-55. The hly promoter and gene fragment were amplified using the primers 5'GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3 '(SEQ ID No: 10; Nhel site underlined) and 5'-CTCCCTCGAGATCATAATTTACTTCATC-3' (SEQ ID No: 11; XhoI site underlined). The prfA gene was amplified in PCR using the primers 5'GACTACAAGGACGATGACCGACAAGTGATAACCCGGGATCTAAATAAATCCGTTT -3 '(SEQ ID No: 12; XbaI site underlined) and 5'-CCCGTCGACC AGCTCTTCTTGGTQAGI No' 13.

[0205] In the resulting plasmid, pGG-55, the hly promoter directs the expression of the first 441 AA of the hly gene product, including the cleaved signal sequence which is attached via the XhoI site to the E7 gene, resulting in the hly-E7 fusion gene, which is transcribed and secreted as 10-E7. This IL10 fragment has no hemolytic C-terminus and has the sequence given in SEQ ID No: 18. This is referred to below as "^ LLO" and is merely an example of Al1010 of many that can be used with the methods and compositions of the invention. Transformation of the prfA-negative Listeria strain, XFl-7 (provided by Dr. Hao Shen, University of Pennsylvania), pGG-55 selected for plasmid preservation in vivo (Figures 1A-B).

[0206] 1m-E7 was generated by introducing an expression cassette containing the hly promoter and signal sequence directing the expression and secretion of E7 into the orfZ domain of the 1M genome. E7 was amplified in PCR using primers 5'GCGGATCCCATGGAGATACACCTAC-3 '(SEQ ID No: 22; BamHI site underlined) and 5'-GCTCTAGATTATGGTTTCTGAG-3' (SEQ ID No: 23; XbaI site underlined). E7

Then ligated into the pZY-21 shuttle vector. The LM 10403S strain was transformed with the resulting plasmid, pZY-21-E7, which contains an expression cassette inserted within 1.6-k of the sequence that corresponds to the orfX, Y, Z domain of the LM genome. The homology domain allows the introduction of the E7 gene cassette into the orfZ domain by homologous recombination. Clones were screened for integration of the E7 gene cassette into the orfZ domain. Bacteria were grown in medium from boiled hearts and brains [BHI] with (Lm-LLO-E7 and Lm-LLO-NP) or without (Lm-E7 and ZY-18) chloramphenicol (20 μg / ml). Bacteria were frozen in portions at -80 ° C. Expression was verified by Western hybridization (Fig. 2)

Western hybridizations [0207] Listeria strains were grown in Luria-Bertoni medium at 37 ° C and harvested at the same optical density measured at 600 nm. Supernatants were TCA precipitated and resuspended in 1x buffer sample with the addition of 0.1 N NaOH. Identical amounts of each cell pellet or each TCA precipitated supernatant were applied to SDS-PAGE - 4-20% Tris-glycine (NOVEX, San Diego, CA) gels. The gels were transferred to polyvinylidene fluoride and probed with anti-E7 monoclonal antibody (mAb) probes (Zymed Laboratories, South San Francisco, CA), followed by incubation with HRP-conjugated anti-mouse secondary Ab (Amersham Pharmacia Biotech, Little Chalfont, UK), developed with Amersham ECL detection reagents and exposed to Hyperfilm (Amersham Pharmacia Biotech).

Tumor growth measurement [0208] Tumors were measured every two days with a caliper covering the shortest and longest surface diameters. The average of these two measurements was plotted as the mean tumor diameter in millimeters at various time points. Mice were sacrificed when the tumor diameter was 20 mm. Tumor measurements for each time point are shown only for surviving mice.

Effect of Listeria recombinants on established tumor growth [0209] Six- to 8-week-old C57BL / 6 mice (Charles River) received 2 x 10⁵TC-1 sc cells in the left side. One week after tumor inoculation, the tumors reached a palpation size of 4-5 mm in diameter. Groups of 8 mice were then treated with 0.1 LD50

ip Lm-LLO-E7 (10⁷ CFU), Lm-E7 (10⁶ CFU), Lm-LLO-NP (10⁷ CFU) or Lm-Gag (5 x 10⁵CFU) on days 7 and 14.

Release designation ⁵¹Cr [0210] C57BL / 6 mice, 6-8 weeks old, were immunized with ip 0.1 LD50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP or Lm-Gag. Ten days after immunization, spleens were harvested. A splenocyte culture was established with exposed TC-1 cells (100: 1, splenocytes: TC-1) as feeder cells; stimulated in vitro for 5 days, then used in a standard release assay⁵¹Cr, using the following targets: EL-4, EL-4 / E7 or EL-4 pulsed with E7 H-2b peptide (RAHYNIVTF).

The E: T cell ratios performed in triplicate were 80: 1, 40: 1, 20: 1, 10: 1, 5: 1, and 2.5: 1. After 4 hours incubation at 37 ° C, cells were pelleted, and 50 ml of supernatant removed from each well. Samples were determined using a Wallac 1450 scintillation counter (Gaithersburg, MD). The percent specific lysis was defined as [(experimental counts per minute - spontaneous counts per minute) / (total counts per minute spontaneous counts per minute)] x 100.

TC-1-specific proliferation [0211] C57BL / 6 mice were immunized with 0.1 LD50 and ip boosted by injection 20 days later with 1 LD50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag . Six days after boost, spleens were collected from immunized and naive mice. Splenocytes were established in 5 x 10 cultures⁵ per well in 96-well flat-bottomed plates with 2.5 x 10⁴, 1.25 x 10⁴, 6 x 10³, or 3 x 10³irradiated TC-1 cells per well - as a source of E7 Ag, or without TC-1 cells or with 10 μg / ml Con A. The cells were pulsed with 45 h. later 0.5 LiCi [³H] thymidine per well. Plates were collected 18 hours. later using a Tomtec 96 harvesting device (Orange, CT), and proliferation was assessed using a Wallac 1450 scintillation counter. Changes in counts per minute were calculated as experimental counts per minute - Ag counts per minute.

Flow cytometry analysis [0212] C57BL / 6 mice were immunized intravenously (iv) with 0.1 LD50 Lm-LLO-E7 or Lm-E7 and boosted 30 days later. Three-color flow cytometry was performed for CD8 (53-6.7, PE conjugated), CD62 ligand (CD62L; MEL-14, APC conjugated), and E7 H-2Db tetramer using a FACSCalibur® flow cytometer with CellQuest® software (Becton Dickinson, Mountain View, CA). Splenocytes harvested 5 days after boosting were stained at room temperature (rt) with H-2Db tetramers loaded with E7 peptide (RAHYNIVTF) or control peptide (HIV-Gag). Tetramers were used at a dilution of 1/200, they were provided by Dr. Larry R. Pease (Mayo Clinic, Rochester, MN) and the National Institute of Allergy and Infectious Diseases Tetramer Core Facility and National Institutes of Health AIDS Research and Reference Reagent Program. Tetramer cells were analyzed⁺, CD8⁺, CD62L^low.

Reduction in the number of specific immune components [0213] The number of CD8 cells was reduced⁺, CD4⁺ and IFN in mice carrying TC-1 by injecting mice with 0.5 mg per mouse mAb: 2.43, GK1.5, or xmg1.2, respectively, on days 6, 7, 8, 10, 12 and 14 after tumor challenge. CD4 cell populations⁺ and CD8⁺decreased by 99% (flow cytometry analysis). The number of CD25 cells⁺ decreased after ip injection of 0.5 mg / mouse anti-CD25 mAb (PC61, provided by Andrew J. Caton) on days 4 and 6. The number of TGF decreased after ip injection of anti-TGF-mAb (2G7, provided by HI Levitsky), in mice carrying TC-1 on days 6, 7, 8, 10, 12, 14, 16, 18 and 20. Mice were treated with⁷ Lm-LLO-E7 or Lm-E7 on day 7 after tumor challenge.

Adoptive transfer [0214] C57BL / 6 donor mice were immunized - and boosted 7 days later with -0.1 LD50 Lm-E7 or Lm-Gag. Donor splenocytes were harvested and passed through nylon wool columns to enrich for T cells. The number of CD8 + T cells decreased in vitro by incubating with 0.1 μg 2.43 anti-CD8 mAb for 30 min at rt. The labeled cells were then treated with rabbit complement system. Donor splenocytes had> 60% CD4 T cells⁺ T (flow cytometry analysis). Mice carrying TC-1 were immunized with 0.1 LD50 7 days after tumor challenge. Enriched in CD4⁺ donor splenocytes (10⁷) was transferred 9 days after tumor challenge to recipient mice by iv injection

B16F0-Ova experiment [0215] 24 C57BL / 6 mice were inoculated with 5 x 10⁵ B16F0-Ova cells. On days 3, 10 and 17, groups of 8 mice were immunized with 0.1 LD50 Lm-OVA (10⁶ cfu), Lm-LLO-OVA (10⁸ cfu) and eight untreated animals were left.

Statistics [0216] For comparisons of tumor diameters, the mean and SD of tumor size were determined for each group, and statistical significance was determined using a student's t test. p <0.05 was considered significant.

RESULTS [0217] Lm-E7 and Lm-LLO-E7 were compared for their ability to affect TC-1 growth. Subcutaneous tumors were established on the left flank of C57BL / 6 mice. Seven days later, the tumors reached palpable size (4-5 mm). Mice were vaccinated on days 7 and 14 with 0.1 LD50 Lm-E7, Lm-LLO-E7 or, as a control, Lm-Gag and Lm-LLO-NP. LmLLO-E7 induced complete regression of 75% of established TC-1 tumors, while two other mice in the group controlled tumor growth (Fig. 3A). In contrast, immunization with Lm-E7 and Lm-Gag did not induce tumor regression. This experiment was repeated many times, always obtaining very similar results. In addition, similar results were obtained for Lm-LLO-E7 with other immunization protocols. In another experiment, a single immunization was able to cure mice from established 5 mm TC-1 tumors.

[0218] In other experiments, similar results were obtained for two other E7-expressing tumor cell lines: C3 and EL-4 / E7. To confirm the efficacy of Lm-LLO-E7 vaccination, tumor removed animals were re-challenged with TC-1 or EL-4 / E7 tumor cells on Day 60 or Day 40, respectively. Animals immunized with Lm-LLO-E7 remained tumor free until the end of the experiment (day 124 for TC-1 and day 54 for EL-4 / E7).

[0219] A similar experiment was performed for chicken ovalbumin antigen (OVA). Mice were immunized with Lm-OVA or Lm-LLO-OVA, then challenged with

- El-4 thymoma expressed to express OVA or the very aggressive mouse B16F0-Ova melanoma cell line, which had very low MHC class I expression. In both cases, lm-lO-OVA but not lm-OVA induced regression of established cancer. For example, after the B16F0 experiment (day 25), all naive and Lm-OVA mice died. All Lm-LLO-OVA mice remained alive, and 50% of them were tumor free. (Fig. 3B).

[0220] Thus, the expression of the antigen gene as an ALLO fusion protein increases the immunogenicity of the antigen.

EXAMPLE 2: TREATMENT LM-LLO-E7 IS IMPORTANT FOR TC-1

SPLENOCITE PROLIFERATION [0221] To measure T-cell induction by Lm-E7, with Lm-LLO-E7, TC-1 specific proliferative responses of splenocytes from rLm immunized mice were assessed - an antigen-specific immunocompetence meter. Splenocytes from Lm-LLOE7 immunized mice proliferate when exposed to exposed TC-1 cells as an E7 source at a splenocyte: TC-1 ratio of 20: 1, 40: 1, 80: 1 and 160: 1 (Fig. 4). Conversely, splenocytes from Lm-E7 immunized and control rLm mice only showed background level proliferation.

EXAMPLE 3: FUSION NP TO LLO INCREASES ITS IMMUNOGENESS

EXPERIMENTAL MATERIALS AND METHODS [0222] Lm-LLO-NP was prepared as shown in Fig. 1, except that influenza nucleoprotein (NP) replaced E7 as an antigen. 32 BALB / c mice were inoculated with 5 x 10⁵ RENCA-NP tumor cells. RENCA-NP means renal cell carcinoma retrovirally transduced with NP nucleoprotein (described in US Patent 5,830,702, which is incorporated herein by reference). After macroscopic palpatory tumor growth on day 10, 8 animals in each group were immunized with 0.1 LD50 ip with the relevant Listeria vector. The animals received a second immunization one week later.

RESULTS [0223] To confirm the universality of the statement that LLO conjugation to antigen confers increased immunity, Lm-LLO-NP and Lm-NP (isogenic with Lm-E7 vectors but expressing influenza antigen) were constructed and vectors were compared for their ability to induce tumor regression, with Lm-Gag (isogenic with Lm-NP except for expressed antigen) as a negative control. As shown in Fig. 5, 6/8 mice that received Lm-LLO-NP were tumor free. In contrast, only 1/8 and 2/8 mice in the Lm-Gag and Lm-NP groups, respectively, were tumor free. All mice in the naive group had large tumors or died before day 40. Hence, LLO strains expressing NP and LLO-NP fusions are immunogenic. Similar results were obtained for Lm-LLO-E7 with other immunization protocols. In addition, it was shown that a single immunization was able to cure mice from established 5 mm TC-1 tumors.

-42 EXAMPLE 4: INCREASING IMMUNOGENESS THROUGH ANTIGEN FUSION

NO LISTERATION VECTOR FOR LLO

EXPERIMENTAL MATERIALS AND METHODS

Construction of Vac-SigE7Lamp [0224] Vaccinia WR strain was used as the recipient, the fusion gene was excised from the Listeria plasmid, and introduced into pSC11 under the control of the p75 promoter. This vector was chosen because it is a transfer vector used in Vaccinia constructs Vac-SigE7Lamp and Vac-E7 and as a consequence will allow direct comparison with Vac-LLO-E7. As a result, all three recombinant vaccinia constructs will be expressed under the control of the same early / late p7.5 promoter compound. In addition, SC11 enables the selection of recombinant viral plaques for selection for TK and beta-galactosidase screening. Fig. 6 shows the different vaccinia constructs used in these experiments. Vac-SigE7Lamp is a recombinant vaccinia virus that expresses the E7 protein coupled between the signal sequence of the lysosome-associated membrane protein (LAMP-1) and the sequence of the LAMP-1 cytoplasmic tail. It has been designed to facilitate antigen targeting on the MHC class II pathway.

[0225] The following modifications were made to allow expression of the gene product in vaccinia: (a) the T5XT sequence that prevents early transcription in vaccinia from the 5 'portion of the LLO-E7 sequence by PCR was removed; and (b) an additional XmaI restriction site was introduced by PCR to allow for the final insertion of LLO-E7 into SC11. Successful introduction of these changes (without losing the original sequence that encodes LLOE7) was verified by sequencing. The resulting construct pSC1 1-E7 was used to transfect the TK-ve CV1 cell line, which was infected with the wild-type vaccinia strain, WR. Cell lysates obtained from this co-infection / transfection step contain a recombinant vaccinia, which has been purified 3 times from plaques. Expression of the LLO-E7 fusion product in plaque purified vaccinia was confirmed by Western blot using an antibody directed against the LLO protein sequence. In addition, Vac-LLO-E7's ability to produce CD8 T cells was determined⁺specific for LLO and E7 using epitopes LLO (91-99) and E7 (49-57) in Balb / c and C57 / BL6 mice, respectively. The results were confirmed in the chromium release assay. RESULTS [0226] To determine whether a Listeria vector was required to increase immunogenicity by antigen fusion to LLO, a vaccinia vector expressing E7 as a fusion protein with a nonhemolytic truncated form of LLO (ALLO) was constructed. Tumor rejection studies for TC-1 were performed using the protocol described in Example 1. Two experiments were performed, with varying delays before starting treatment. In one experiment, treatment was initiated when the tumors were about 3 mm in diameter (Fig. 7). As of Day 76, 50% of Vac-LLO-E7-treated mice were tumor free, while only 25% of Vac-SigE7Lamp mice were tumor free. In other experiments, ALLO-antigen fusions were more immunogenic than the E7 peptide

Mixed with SBAS2 or unmethylated CpG oligonucleotides in one-second comparison.

[0227] These results show that (a) ΔLLO-antigen fusions are immunogenic not only in the context of Listeria, but also in other contexts; and (b) the immunogenicity of the LLOantigen fusion preferably withstands comparison with other acceptable vaccine-type approaches.

EXAMPLE 5: ACTA-ANTIGEN AND PEST-ANTIGENE FUSES GIVE RESISTANCE

antineoplastic

EXPERIMENTAL MATERIALS AND METHODS

Construction of Lm-PEST-E7, Lm- \ PEST-E7 and Lm-E7epi (Fig. 8A) [0228] Lm-PEST-E7 is identical to Lm-LLO-E7, except that it only contains the promoter and the PEST sequence hly gene, more specifically the first 50 AA LLO. To construct Lm-PEST-E7, the hly promoter and PEST region were coupled to the full-length E7 gene using PCR - SOE (gene splicing by lengthening pleats). The E7 gene and hly-PEST gene fragment from plasmid pGG-55, which contains the first 441 AA LLO, were amplified and spliced ​​by conventional PCR techniques. To create the final plasmid, pVS16.5, the hly-PEST-E7 fragment and the prfA gene were subcloned into the pAM401 plasmid, which contains the chloramphenicol resistance gene for in vitro selection, and the resulting plasmid was used for XFL-7 transformation.

[0229] Lm- \ PEST-E7 is a recombinant Listeria strain that is identical to LmLLO-E7 except that it lacks the PEST sequence. It was obtained essentially as described for Lm-PEST-E7, except that the episomal expression system was constructed using primers designed to remove the PEST containing region (pz 333-387) from the hly-E7 fusion gene. Lm-E7epi means a recombinant strain that secretes E7 without the PEST or LLO region. The plasmid used to transform this strain contains the hly promoter gene fragment and the signal sequence coupled to the E7 gene. This construct differs from the original Lm-E7, which expressed a single copy of the E7 gene integrated into the chromosome. Lm-E7epi is completely isogenic to Lm-LLO-E7, Lm-PEST-E7 and Lm- \ PEST-E7 except for the expressed E7 antigen.

Construction of Lm-actA-E7 [0230] Lm-actA-E7 means a recombinant LM strain containing a plasmid that expresses the E7 protein coupled to the truncated version of the actA protein. Lm-actA-E7 was generated by inserting plasmid vector pDD-1 constructed by modifying pDP-2028 into LM. pDD-1 contains an expression cassette expressing a 310 bp copy of the hly promoter and hly (ss) signal sequence that directs the expression and secretion of actA-E7; 1170 bp of the actA gene, which contains 4 PEST sequences (SEQ ID No: 16) (the shortened ActA polypeptide consists of the first 390 AA molecules, SEQ ID No: 15); 300 bp of the HPV E7 * gene; 1019 bp

PrfA * gene (controls expression of virulence genes); and the CAT gene (chloramphenicol resistance gene) for selecting transformed bacterial clones. (Fig. 8B).

[0231] The hly promoter (pHly) and gene fragment were PCR amplified from pGG-55 (Example 1) using primers

5'-GGGGTCTAGACCTCCTTTGATT AGT AT ATTC-3 '(site Xba I underlined; SEQ ID NO: 46) and 5'-ATCTTCGCTATCTGTCGCCGCGGCGCGTGCTTCAGTTTGTTGCGC-'3 (site Not I highlighted; first 18 nucleotides of ID NO: 47 . actA gene was PCR amplified from wild-type LM 10403s using the primer 5 '-GCGC AAC AAACTGAAGC AGCGGCCGCGGCGACAGATAGCGAAGAT-3' (NotI site underlined; SEQ ID NO: 48) and primer 5'TGTAGGTGTATAGATCATGTCTC ID NO: 49). the E7 gene was PCR amplified from pGG55 (pLLO-E7) using the 5'-GGAATTGATCGCCTAGCTCTCGAGCATGGAGAGACACACCTACA-3 'primer (XhoI site underlined; SEQ ID NO: 50) and the 5AAACGGATTTATATTTATTGATGGTGATTGATA 51). the prfA gene was PCR amplified from LM 10403s wild-type genome using a primer

5'-TGTTCTCAGAAACCATAACCCGGGATCTAAATAAATCCGTTT-3 '(XmaI site underlined; SEQ ID NO: 52) and the primer 5'-GGGGGTCGACCAGCTCTTCTTGGTGAAG-3' (SalI site underlined; SEQ ID NO: 53). The hly promoter coupled to the actA gene (pHlyactA) was generated by PCR and amplified from purified pHly DNA, and actA DNA purified using the upstream pHly primer (SEQ ID NO: 46) and the downstream actA primer (SEQ ID NO: 49 ).

[0232] The E7 gene coupled to the prfA gene (E7-prfA) was generated by PCR and amplified from purified E7 DNA and purified prfA DNA using the upstream E7 primer (SEQ ID NO: 50) and the downstream prfA gene ( SEQ ID NO: 53).

[0233] The pHly-actA fusion product coupled to the E7-prfA fusion product was generated by PCR and amplified from the purified DNA conjugate product pHly-actA, and the coupled DNA product E7-prfA was purified using the upstream pHly primer (SEQ ID NO: 46) and the underlying prfA gene primer (SEQ ID NO: 53) and ligated into pCRII (Invitrogen, La Jolla, Calif.). Competent E. coli (TOP10'F, Invitrogen, La Jolla, Calif.) Were transformed with pCRII-ActAE7. After lysis and isolation, the plasmid was screened by restriction analysis using BamHI (expected fragment sizes of 770 bp and 6400 bp (or when the insert was reversed in the vector: 2500 bp and 4100 bp)) and BstXI (expected fragment sizes 2800 bp and 3900 pz) and also screened by PCR analysis using the upstream pHly primer (SEQ ID NO: 46) and the downstream prfA gene primer (SEQ ID NO: 53).

[0234] The pHly-ActA-E7-PrfA DNA insert was excised from pCRII by double digestion with Xba I and Sal I and ligated into pDP-2028 also digested with Xba I and Sal I. After transformation

TOP10'F competent E. coli (Invitrogen, La Jolla, Calif.) PActAE7 expression system, chloramphenicol resistant clones were screened by PCR analysis using the pHly primer located above (SEQ ID NO: 46) and below the PrfA gene primer (SEQ ID NO: 53). A pHly-ActA-E7-bearing clone was cultured in medium from boiled hearts and brains with 20 mcg (micrograms) / ml (milliliter) chloramphenicol (Difco, Detroit, MI), and pActAE7 was isolated from bacterial cells using a midiprep DNA purification system kit (Promega, Madison, Wis.). The penicillin treated strain of Listeria XFL-7 was transformed with pActAE7, and clones were selected for in vivo plasmid retention. Clones were cultured in medium from boiled hearts and brains, with chloramphenicol (20 mcg / ml) at 37 ° C. Bacteria were frozen in portions at -80 ° C. RESULTS [0235] To compare the anti-tumor immunity induced by Lm-ActA-E7 vs. Lm-LLO-E7, 2 x 10⁵ TC-1 tumor cells were implanted subcutaneously in mice and allowed to grow to a palpation size (approximately 5 millimeters [mm]). Mice were immunized with one LD50 Lm-ActA-E7 (5 x 10)⁸ CFU), (crosses) Lm-LLO-E7 (10⁸ CFU) (squares) or Lm-E7 (10⁶ CFU) (circles) on days 7 and 14. Until day 26, all of the animals in Lm-LLO-E7 and Lm-ActA-E7 were tumor-free and remained so, while in all naive animals (triangles) and immunized animals Lm-E7 developed large tumors (Fig. 9). Therefore, vaccination with ActA-E7 fusions causes tumor regression.

[0236] In addition, Lm-LLO-E7, Lm-PEST-E7, 1.m-APP.ST-P7 and Lm-E7epi were compared for their ability to cause regression of E7 expressing tumors. TC-1 tumors were established on the left flank of 40 C57B1 / 6 mice. After the tumors reached 4-5 mm, the mice were divided into 5 groups of 8 mice. Each group was treated with 1 out of 4 recombinant 1M vaccines and 1 group was left untreated. 1m-11O-E7 and 1m-PEST-E7 induced regression of established tumors in 5/8 and 3/8 cases, respectively. There were no statistical differences between the mean tumor size of mice treated with 1m-PEST-E7 or 1m-11O-E7 at any time point. However, vaccines that expressed E7 without the PEST sequence, 1.m-APP.ST-P7 and 1m-E7epi, failed to cause tumor regression in all but one of the mice (Fig. 8C). This was representative of two experiments, with a statistically significant difference in mean tumor sizes on day 28 between tumors treated with 1m-11O-E7 or 1m-PEST-E7 and those treated with 1m-E7epi or 1m-APP.ST- P7; p <0.001, student t-test; Fig. 8D). In addition, increased percentages of tetramer-positive splenocytes were repeated, repeatedly, in three experiments in the spleen of mice vaccinated with vaccines containing PEST (Fig. 8E). Hence vaccination with PEST-E7 fusions causes tumor regression.

EXAMPLE 6: FUSION E7 TO LLO, ACTA OR SEQUENCE SEQUENCE

INCREASES IMMEDIATE RESISTANCE FOR ANTIGENE AND GENERATES INFILTRATING CANCER CD8 + CELLS SPECIAL FOR E7 EXPERIMENTAL MATERIALS AND WAYS

[0237] 500 mcl (microliters) MATRIGEL®, containing 100 mcl 2 x10⁵ TC-1 tumor cells in phosphate buffered saline (PBS) plus 400 mcl MATRIGEL® (BD Biosciences, Franklin Lakes, NJ) were implanted subcutaneously in the left flank of 12 C57BL / 6 mice (n = 3). Mice were immunized intraperitoneally on days 7, 14 and 21, and spleens and tumors were collected on day 28. Tumor MATRIGELs were removed from the mice and incubated at 4 ° C overnight in tubes containing 2 milliliters (ml) of RP 10 medium on ice. Tumors were minced with tweezers, cut into 2 mm blocks and incubated at 37 ° C for 1 hour with 3 ml enzyme mixture (0.2 mg / ml collagenase-P, 1 mg / ml DNAse-1 in PBS). The tissue suspension was filtered through a nylon screen and washed with 5% fetal bovine serum + 0.05% NaN3 in PBS for staining tetramers and IFN-gamma.

[0238] Splenocytes and tumor cells were incubated with 1 micromole (mcm) of E7 peptide for 5 hours in the presence of brefeldine A at 10⁷ cells / ml. Cells were washed twice and incubated in 50 mcl anti-mouse Fc receptor supernatant (2.4 G2) for 1 hour or overnight at 4 ° C. Cells were stained for surface CD8 and CD62L molecules, permeabilized, fixed using the Golgistop® or Golgi-Plug® permeabilization kit (Pharmingen, San Diego, Calif.) And stained for IFN-gamma. 500,000 events were obtained using a flow cytometer with two FACSCalibur lasers and analyzed using Cellquest software (Becton Dickinson, Franklin Lakes, NJ). The percentage of IFN-gamma secreting cells within activated cells (CD62Llow) T CD8 was calculated⁺.

[0239] With respect to tetramer staining, H-2D tetramer^b loaded with phycoerythrin (PE) coupled to peptide E7 (RAHYNIVTF, SEQ ID NO: 24), stained at rt for 1 hour, and stained with anti-allophycocyanin (APC) coupled with MEL-14 (CD62L) and FITC coupled to 4 ° C for 30 min. Cells were analyzed by comparing tetramer cells⁺CD8⁺ CD62L^low in the spleen and in cancer.

RESULTS [0240] To analyze the ability of Lm-ActA-E7 to increase antigen-specific immunity, TC-1 tumor cells were implanted in mice and immunized with Lm-LLO-E7 (1x10⁷ CFU), Lm-E7 (1 x 10⁶ CFU) or Lm-ActA-E7 (2 x 10⁸ CFU) or left untreated (naive). Mouse tumors of the Lm-LLO-E7 and Lm-ActA-E7 groups contained a higher percentage of IFN-gamma secreting CD8 T cells⁺ (Fig. 10A) and CD8 + tetramer specific cells (Fig. 10B) than in Lm-E7 or naive mice.

[0241] In a further experiment, tumor bearing mice were administered Lm-LLO-E7, Lm-PEST-E7, 1.m-APHST-H7 or Lm-E7epi, and the levels of E7-specific lymphocytes within the tumor were measured. Mice were treated on days 7 and 14 with 0.1 LD50 of four vaccines. Tumors were harvested on day 21 and stained with antibodies to CD62L, CD8 and E7 / Db tetramer. An increased proportion of tetramer positive lymphocytes within the tumor was observed in Lm-LLO-E7 and Lm-PEST-E7 vaccinated mice (Fig. 11A). This result was reproducible in three experiments (Fig. 11B).

[0242] Hence, Lm-LLO-E7, Lm-ActA-E7 and Lm-PEST-E7 are each of them effective at inducing CD8 T cells⁺ cancer infiltrating and tumor regression.

EXAMPLE 7: CREATING AND VERIFYING LISTERIA-LLOPS CONSTRUCTIONS

EXPERIMENTAL MATERIALS AND METHODS [0243] Oligos and pCDNA3.1 were purchased from Invitrogen (Carlsbad, CA) and DNA sequencing was performed at Genewiz Inc. (Plainfield, NJ). Peptides were synthesized by EZbiolabs (Westfield, IN). Flow cytometry reagents were purchased from Becton Dickinson (BD) Biosciences (San Diego, CA). Cell culture media and additives were from Gibco / Invitrogen. ELISpot antibodies were purchased from Mabtech AB (Cincinnati, OH). Other reagents, unless otherwise indicated, were from Sigma (St. Louis, MO). PSA / vaccinia were obtained courtesy of Dr. Schlom of the National Cancer Institute, Bethesda, MD.

Mice, cell lines and media [0244] C57BL / 6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were kept at the Cook Campus in animal housing, at Rutgers University, New Brunswick, NJ. Experiments on mice were performed according to the regulations of the Institutional Animal Care and Use Committee of Rutgers University. All cell lines were purchased from ATCC (Manassas, VA). The TRAMPC-1 (Transgenic Adenocarcinoma of the Mouse Prostate) cell line; transgenic mouse prostate adenocarcinoma) derived from C57BL / 6 mice has previously been described. Cells were cultured and maintained in modified Dulbecco's Eagle medium with 4 mM L-glutamine, adapted to contain 1.5 g / L sodium bicarbonate and 4.5 g / L glucose with the addition of 5 μg / ml bovine insulin, 10 nM dehydroisoisoandrosterone, 5% fetal bovine serum and 5% Nu-Serum IV. MC57G fibrosarcoma cells (C57BL / 6 background) were maintained in Eagle's minimal nutrient medium (EMEM) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g / L sodium bicarbonate, 0.1 mM essential amino acids, 1 , 0 mM sodium pyruvate and 10% fetal bovine serum. EL4 lymphoma cells were maintained in modified Dulbecco's Eagle medium with 4 mM L-glutamine, adjusted to contain 1.5 g / L sodium bicarbonate and 4.5 g / L glucose supplemented with 10% fetal bovine serum. J774A.1 murine macrophage cell cells were maintained in RPMI1640 medium with 4 mM L-glutamine, adjusted to contain 1.5 g / L sodium bicarbonate and 4.5 g / L glucose supplemented with 10% fetal bovine serum. RPMI complete medium (C-RPMI) contained RPMI 1640 with the addition of 2 mM glutamine, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% fetal bovine serum.

Construction of the PSA / pCDNA3.1 vaccine [0245] The entire PSA open reading frame including its signal sequence was cloned into the pCDNA3.1 (-) vector backbone (Invitrogen) at XbaI and XhoI sites. In this plasmid, PSA expression was under the control of the CMV promoter and confirmed by transient

Transfection into 293 cell line (ATCC). Secretion of PSA by transfected cells was confirmed in the culture cell supernatant using an anti-PSA ELISA kit (American Qualex, San Clemente, CA). The GM-CSF / pCDNA plasmid was obtained courtesy of Dr. Vonderheide, University of Pennsylvania, Philadelphia, PA.

Vaccine construction / u - // O-PSA [0246] The gene encoding human PSA (except for its secretory signal sequence) was amplified using pfx polymerase, a sense oligo:

gtgCTCGAGattgtgggaggctgggagtg (SEQ ID NO: 58) and antisense oligo: gatACTAGTttaggggttggccacgatgg (SEQ ID NO: 59). The two restriction sites, XhoI and Spel, which were used to clone PSA in the reading frame as a fusion to LLO in pGG55 are indicated in capital letters. The stop codon to complete translation of the LLOPSA fusion protein is underlined in the antisense oligo. The amplified PSA was cloned into the Lm plasmid pGG55 as a fusion protein to the first 441 amino acids at the N terminus of LLO (pAdv34, Fig. 12), between XhoI and SpeI restriction sites. The resulting plasmid was sequenced to confirm no mutation. Plasmid pAdv34 was electropolated to the Lm XFL7 strain (which lacks the genomic prfA gene) and was first obtained from streptomycin resistant strain Lm 10403s. Plasmid-containing listeriae (Lm-LLO-PSA) was selected using chloramphenicol (34 μg / ml) and 250 μg / ml streptomycin in dishes with medium from boiled hearts and brains (BHI / Chl / S). Colonies were screened by PCR to confirm the presence of the PSA gene.

Subcloning // O-PSA [0247] The truncated PSA open reading frame (GenBank accession number NM_001648), lacking the secretory signal sequence, the first 24 AA, was amplified using the primers: Adv60-PSA (XhoI-no ATG) F: gtgCTCGAGattgtgggaggctgggagtg (SE No: 58) and Adv61-PSA (SpeI-Stop) R: gatACTAGTttaggggttggccacgatgg (SEQ ID No: 59) and subclones, in reading frame, with the first 441 amino acids of LLO (Fig. 12). The plasmid skeleton pGG55 (Example 1) also has a copy of the Listeria prfA virulence gene, and two chloramphenicol acetyltransferase genes that confer chloramphenicol resistance to both gram-positive and gram-negative bacterial strains. The AA LLO-PSA sequence is as follows:

-49MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQ

GLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYP

GALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVER

WNEKYAQAYPNVSAKIDYDDEMAYSESQL1AKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS

FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHST

KVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATF

NRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVN

YDLEIVGGWECEKHSQPWOVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLF

HPEDTGOVFOVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPT

QEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAOVHPQKVTKFMLCAGRWTG

GKSTCSGDSGGPLVCYGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID (SEQ ID No: 54; PSA sequence underlined).

[0248] There is one AA difference between this PSA and the sequence at NM_001648, at position N 221 Y). pGG55-LLO-PSA was electropolated into L. monocytogenes XFL-7 (Example

1).

Growth and storage of bacterial vaccine strains [0249] Recombinant Listeria-PSA was cultured in animal-free medium (modified Terrific Broth) in the presence of 34 μg / ml chloramphenicol and 250 μg / ml streptomycin, at 37 ° C in a thermostat with shaker. After reaching an optical density (OD600) of 0.5, which indicated a logarithmic growth phase, the bacteria were collected by centrifugation, and the pellet was washed twice in phosphate buffered saline (PBS) and resuspended in PBS containing 2% glycerol, then aliquoted and stored at -80 ° C. One aliquot was thawed 1 day later and titrated to determine bacterial titer (colony forming units / ml). So stored Listeria vaccines are stable for up to 1 year. These portions were then thawed, diluted to 1x10⁷ CFU per dose and used in immunogenicity studies as follows.

Expression of LLO-PSA for Lm-LLO-PSA [0250] Lm-LLO-PSA colonies were grown in BHI / Chl / S broth at 37 ° C and 225 rpm for 8 hours. Bacteria were separated from the culture medium by centrifugation at 10,000 g for 5 min and supernatants were recovered. Proteins in culture supernatants were precipitated for at least 1 hour in the presence of 10% v / v. trichloroacetic acid at 4 ° C and separated on a 420% gel by SDS-PAGE. After transferring the proteins to PVDF membranes, the LLO-PSA fusion protein was detected by immunological hybridization with rabbit polyclonal anti-LLO (prepared on site) or anti-PSA antibodies (Sigma). Signals were developed using a Western-Breeze chromogenic Kit (Invitrogen). In vivo passaging of Lm-LLO-PSA.

[0251] Lm-LLO-PSA was passaged in vivo twice. Briefly, 1 x 10 dose⁶ CFU LmLLO-PSA was injected intraperitoneally (ip) into 6-8 week old mice. Spleens were harvested 3 days after injection; single cell suspensions were obtained from spleens and poured onto BHI / Chl / S dishes for Lm-LLO-PSA growth selection. This procedure was repeated one more time using the LLO-PSA positive colony from the first passage. Lm-LLO-PSA colonies recovered after two in vivo passages were tested for LLO-PSA secretion. A single clone was selected for use for plasmid DNA sequencing, cancer research and other functional assays.

In vivo virulence study [0252] Virulence studies were performed to determine the safe dose of Lm-LLO-PSA in mice. Two groups of 4 male C57BL / 6 mice (7 weeks old) were injected with two different doses of Lm-PSA: 1x10⁸ and 5 x 10⁷ CFU per dose. Mice were monitored for signs of disease up to 5 days after inoculation.

Plasmid sequencing [0253] Lm-LLO-PSA was grown overnight in 200 ml BHI / Chl / S at 37 ° C and at 225 rpm. Bacteria were separated from the culture broth by centrifugation at 10,000 g at 4 ° C for 10 min. The bacterial pellet was treated with lysozyme (2 mg / ml) at 37 ° C for 30 min to disrupt cell walls. Plasmid pAdv34 was purified using the PureLink midiprep purification kit (Invitrogen) according to the manufacturer's instructions and sequenced entirely by dideoxy [chain termination].

RESULTS [0254] A Listeria strain was created that expresses nonhemolytic LLO conjugated to truncated PSA (kallikrein-related peptidase 3). The resulting recombinant Listeria strain secretes a protein size predicted for LLO-PSA (75 Kd) that is detected by both anti-LLO and anti-PSA antibodies, which shows that the LLO-PSA protein was expressed and secreted (Fig. 13).

[0255] Generation of recombinant L. monocytogenes expressing PSA. The gene coding for human PSA was cloned as a fusion protein with a truncated, nonhemolytic LLO segment that contains a conserved PEST sequence at its N-terminus (Fig. 12). The plasmid backbone pGG55 as previously described allows the expression and secretion of LLO fusion proteins from the episomal initiation site by the attenuated Lm XFL7 strain, which lacks the prfA genomic virulence factor. The prfA gene is required for Lm survival in vivo and is therefore complemented by a copy found on the plasmid. The PSA signal sequence was removed to avoid any interaction of this sequence with the correct secretion of the LLO-PSA fusion protein mediated by the LLO secretion signal sequence in Lm-LLO-PSA.

[0256] Lm-LLO-PSA can express and secrete an LLO-PSA fusion protein. Expression and secretion of the LLO-PSA fusion protein from Lm was tested in culture supernatants

-51 cells after in vitro growth of bacteria. Four Lm-LLO-PSA colonies were tested and all were able to secrete LLO-PSA fusion protein as detected by both anti-LLO and anti-PSA antibodies (Fig. 13). This secretion was maintained in bacteria isolated after two in vivo passages in mice (data not shown), suggesting that the plasmid is stable and preserved in vivo for at least three days. To confirm stability, pAdv34 was extracted from passaged LmLLO-PSA and fully sequenced. No mutations were found in the plasmid backbone or the PSA gene after two passages in vivo compared to the reference plasmid pGG55 and the human PSA gene sequence (NCBI: NM_001648). To determine the safe dose of LmLLO-PSA for use in animal studies, two doses of this construct were tested in mice by ip injection (1 x 10 and 5 x 10 CFU per dose). The dose used in₇ animal experiments (1x10 CFU / mouse) were defined as one-tenth of the lowest dose at which it was observed to have a detrimental effect on immunized mice.

[0257] To test in vitro stability of Lm-PSA, the strain was cultured and passaged for 7 consecutive days in modified terrific broth. After this time, the bacteria retained the plasmid, the plasmid contained the PSA gene and no deletions or rearrangements were found in the plasmid, indicating the plasmid stability (Fig. 22).

[0258] To test the in vivo stability of Lm-PSA, the strain was passaged twice in mice. The plasmid was then sequenced with Genewiz ™ and found to have the following sequence:

AATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTT

GTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTA

TAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGAT

ATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAA

ATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGG

AACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGT

ATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTAT

TCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTT

TGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGG

-52TGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTT

ACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGG

CTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTG

ACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGA

GATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAG

CCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAG

TGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTC

GTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTT

GTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTAT

GCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC

AACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGG

AGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACT

GCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAA

AACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAAC

GATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGCCCTCCTTTGATTAGTA

TATTCCTATCTTAAAGTTACTTTTATGTGGAGGCATTAACATTTGTTAATGACGTCAAAAG

GATAGCAAGACTAGAATAAAGCTATAAAGCAAGCATATAATATTGCGTTTCATCTTTAGA

AGCGAATTTCGCCAATATTATAATTATCAAAAGAGAGGGGTGGCAAACGGTATTTGGCAT

TATTAGGTTAAAAAATGTAGAAGGAGAGTGAAACCCATGAAAAAAATAATGCTAGTTTTT

ATTACACTTATATTAGTTAGTCTACCAATTGCGCAACAAACTGAAGCAAAGGATGCATCT

GCATTCAATAAAGAAAATTCAATTTCATCCATGGCACCACCAGCATCTCCGCCTGCAAGT

CCTAAGACGCCAATCGAAAAGAAACACGCGGATGAAATCGATAAGTATATACAAGGATT

GGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGC

CAAGAAAAGGTTACAAAGATGGAAATGAATATATTGTTGTGGAGAAAAAGAAGAAATCC

ATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCAATTTCGAGCCTAACCTATCCA

GGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAATCAACCAGATGTTCTCCCTGTA

AAACGTGATTCATTAACACTCAGCATTGATTTGCCAGGTATGACTAATCAAGACAATAAA

ATAGTTGTAAAAAATGCCACTAAATCAAACGTTAACAACGCAGTAAATACATTAGTGGA

AAGATGGAATGAAAAATATGCTCAAGCTTATCCAAATGTAAGTGCAAAAATTGATTATGA

TGACGAAATGGCTTACAGTGAATCACAATTAATTGCGAAATTTGGTACAGCATTTAAAGC

TGTAAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAG

AAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTT

CCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAG

AAAATCCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGTCAAGTTTATTTGAAATTATC

AACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGATGCTGCCGTAAGCGGAAAATC

TGTCTCAGGTGATGTAGAACTAACAAATATCATCAAAAATTCTTCCTTCAAAGCCGTAAT

TTACGGAGGTTCCGCAAAAGATGAAGTTCAAATCATCGACGGCAACCTCGGAGACTTACG

-53CGATATTTTGAAAAAAGGCGCTACTTTTAATCGAGAAACACCAGGAGTTCCCATTGCTTA

TACAACAAACTTCCTAAAAGACAATGAATTAGCTGTTATTAAAAACAACTCAGAATATAT

TGAAACAACTTCAAAAGCTTATACAGATGGAAAAATTAACATCGATCACTCTGGAGGATA

CGTTGCTCAATTCAACATTTCTTGGGATGAAGTAAATTATGATCTCGAGattgtgggaggctgggagt gcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtoctcacag ctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcctgtttcatectgaagacacaggccaggtatttcaggtcagccacag cttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcag agcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggca gcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaagg tgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgttatggtgtgcttcaa ggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacacc atcgtggccaaccccTAAACTAGTGACTACAAGGACGATGACGACAAGTGATACCCGGGATCTAA

ATAAATCCGTTTTTAAATATGTATGCATTTCTTTTGCGAAATCAAAATTTGTATAATAAAA

TCCTATATGTAAAAAACATCATTTAGCGTGACTTTCTTTCAACAGCTAACAATTGTTGTTA

CTGCCTAATGTTTTTAGGGTATTTTAAAAAAGGGCGATAAAAAACGATTGGGGGATGAGA

CATGAACGCTCAAGCAGAAGAATTCAAAAAATATTTAGAAACTAACGGGATAAAACCAA

AACAATTTCATAAAAAAGAACTTATTTTTAACCAATGGGATCCACAAGAATATTGTATTT

TCCTATATGATGGTATCACAAAGCTCACGAGTATTAGCGAGAACGGGACCATCATGAATT

TACAATACTACAAAGGGGCTTTCGTTATAATGTCTGGCTTTATTGATACAGAAACATCGG

TTGGCTATTATAATTTAGAAGTCATTAGCGAGCAGGCTACCGCATACGTTATCAAAATAA

AGGAACTAAAAGAACTACTGAGCAAAAATCTTACGCACTTTTTCTATGTTTTCCAAACCCT

ACAAAAACAAGTTTCATACAGCCTAGCTAAATTTAATGATTTTTCGATTAACGGGAAGCT

TGGCTCTATTTGCGGTCAACTTTTAATCCTGACCTATGTGTATGGTAAAGAAACTCCTGAT

GGCATCAAGATTACACTGGATAATTTAACAATGCAGGAGTTAGGATATTCAAGTGGCATC

<img file="PL2155243T4_D0001.tif" />

TATAAAAATTCATGCTTTTATGTACAAAATCGTGATTATCTCAAAAGATATGCCCCTAAAT

TAGATGAATGGTTTTATTTAGCATGTCCTGCTACTTGGGGAAAATTAAATTAAATCAAAA

ACAGTATTCCTCAATGAGGAATACTGTTTTATATTTTATTCGAATAAAGAACTTACAGAA

GCATTTTCATGAACGCGTACGATTGCTTCACCAAGAAGAGCTGGTCGACCGATGCCCTTG

AGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCA

CTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCA

TTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTAT

TCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGG

CGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGC

GTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATC

GGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACA

GCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTC

-54ACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCC

GCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCG

ACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCC

AATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATC

GCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGCTTTCGATTTGTTTTTGAATG

GTTTATCCGATAAAGAAGTTGAAGAACAAACTGGAATCAATCGCCGAACGTTTAGAAGG

TATCGAGCAAGATATAACGTGACAGTCGATCAAAGAAAAAACAATGAAAAGAGGGATAG

TTAATGAGTACGGTTATTTTAGCTGAAAAACCAAGCCAGGCATTAGCCTACGCAAGTGCT

TTAAAACAAAGCACCAAAAAAGACGGTTATTTTGAGATCAAAGACCCACTATTTACAGAT

GAAACGTTTATCACCTTTGGTTTTGGGCATTTAGTGGAATTAGCAGAACCAGGTCATTATG

ACGAAAAGTGGCAAAATTGGAAACTTGAATCTTTGCCGATTTTTCCTGATCGATACGATT

TTGAAGTTGCAAAAGATAAGGGAAAGCAGTTTAAAATTGTTGCAGAACTTCTCAAAAAG

GCAAATACAATTATTGTTGCAACAGATAGCGACAGAGAAGGTGAAAATATCGCCTGGTC

GATTATCCATAAAGCAAATGCCTTTTCAAAAGATAAAACATTTAAAAGACTATGGATCAA

TAGCTTAGAAAAAGATGTAATCCGAAGCGGTTTTCAAAATTTGCAACCTGGAATGAATTA

CTATCCCTTTTATCAAGAAGCGCAAACACGCCAAATTGCCGATTGGTTGATCGGCATGAA

CGCAAGCCCTTTGTATACGTTAAATTTACAACAGAAGGGCGTACAAGGTACATTTTCACT

AGGACGTGTTCAAACGCCCACCTTATACCTTATTTTTCAGCGCCAGGAAGCCATAGAGAA

TTTTAAAAAAGAACCTTTTTTCGAGGTGGAAGCTAGTATAAAAGTAAACCAAGGGTCGTT

TAAGGGCGTTCTAAGCCCCACACAGCGTTTTAAAACCCAAGAGGAGCTTTTAGCTTTTGT

TTCTTCTAAACAAGCTAAAATAGGCAATCAAGAGGGGATAATTGCTGATGTTCAAACCAA

AGAGAAGAAAACGAATAGTCCGAGTTTGTTTTCTTTAAGTAGTTTGCAATCAAAAGTCAA

TCAGCTTTATAAAGCGACAGCGAGCCAAACTTTAAAAGCTATTTCTTTTTTAATAACTTAA

AAATAAACTTAATGTAACAGCAAGCACAGTCAAGGTATACACCTTTGACAAAAAATAGC

ACATTCTCTATCGAAAATTTTTGCTTATTTTTTAAATTATTTTGGGAAATTTTCCCAATCCC

TTTTTCTAACTCAAAAAATATAATCACTCAAAATTTAAAAGGGCGCACTTATACATCATTT

TAAAAAATTGATGTAACGTGCTAAGTTCAAAACAAAGGGCGCACTTATACACGATTTTCA

ATCTTGTATATTTCTAACGAAAAGCGTGCGCCAAAAAACCCCCTTCGTCAATTTTGACAG

GGGGCTTTTTGATGTAAAAATTTCTATCGAAATTTAAAAATTCGCTTCACTCATGTTATAA

AGACTTAAAATAAAATAACTCTTTAAAATCTTTTGCTAGTTGTTCTTCAATATTTTTTATTC

GGTGCATCTTCCAAGTAAAGTATAACACACTAGACTTATTTACTACGTTTCATAAGTCATT

AATGCGTGTGCTCTGCGAGGCTAGTTTTTGTGCAAGCACAAAAAATGGACTGAATAAATC

AGTCCATAAGTTCAAAACCAAATTCAAAATCAAAACCACAAGCAACCAAAAAATGTGGT

TGTTATACGTTCATAAATTTTATGATCACTTACGTGTATAAAATTAAATTCACTTTCAAAA

TCTAAAAACTAAATCCAATCATCTACCCTATGAATTATATCTTGAAATTCATTCATAAATA

GTGAAGCATGGTAACCATCACATACAGAATGATGAAGTTGCAGAGCAACTGGTATATAA

ATTTTATTATTCTCACTATAAAATTTACCTATCGTAATAATAGGCAATAAAAAGCTGCTAT

-55TGTTACCAATATTTAAATTAAATGAACTAAAATCAATCCAAGGAATCATTGAAATCGGTA

TGGTGTTTTCAGGTATCGGTTTTTTAGGAAACATTTCTTCTTTATCTTTATATTCAAGCAAG

TAAATTTTTCAGTTTGCTTATTAAAAACTGTATACAAAGGATTTAACTTATCCCAATAACC

TAATTTATTCTCACTATTAATTCCTGTTCTAAACACTTTATTTTTATTTACAACTTCCATAA

TTGCATAAATTAAAGAGGGATAAATTTCATATCCTTTCTTTTTTATCATATCTTTAAACAA

AGTAATATCAATTTCTTTAGTAATGCTATAAGTAGTTTGCTGATTAAAATAGTGTTCAAAA

TATTCTTTTCTATCCCAATTTTCTAATTCAATAATATTAAAAGTCATATATAACTTCCTCCT

AAATTTTAAATTTTTATATTTAGGAGGAATAATCCTCTGATTTTTTCATACGTTATGTCACC

TCGTAAATATTAATTATACTGAATTAGCAATTTTTATCAAATAAAACTTATTTTACTTCCA

AAACCTAAATTCACGTTGCCAAAAATCAATCTGCTTTTGCAATTGTTTTTCGTTCGCTTTT

AAAGTCGATTTCATTAATTCCGTTAAATCAATTGGAGATATTTCTCTAATCAATTTTTTAA

ATTTAGTCTTAGTATTCTTACTTAGCTTTCCCCACATACTTTCTTCATGCAACAAAGTATAA

ACCATAGCTTGCTCATTAATTTTTTCTAAAGTAGCCCACGCAGGTTTCAAGATGTGTAAAT

TAAATGTTCAGACACAACTTCAACATCTGCGTTATCTTTACGTTCTTGTTTTTTATTATAAA

TTCTAATAAATCTATCACTATCACGGACACCAAAATATTTTGTTTCTGGCTTGCCATTACG

ACCATAAAAAACAGTTTTCTTAACTGCTTTATCAGTCATTGCATAGTAATCGCTCAAATCA

AAAA Τ'Ζ'Ί AAAA Ζ-ΙΖΊΤ · AAZ ^ T ^ Z ^ T ^ AAAAAA Z '' / - 'Z'^-'"Τ'Z' 'A' ΤΓ'Τ'ΓΖ¹/ "'A ΠΓΖΊ'Τ^-'A ΛΤΛ Λ

ΙΌ 1 1 IV / Y / Y / Y / YYJL · * 1 ΛΛΌ 1Ό1 ΖΥ2Υ 1 ^ 1 1 YJ and ΛΛΛΛΌΥΌ 1 Vz / Y 1 Ό ί 1 1 YJ 1 / YVJ I 1 ΖΎΑΎ

TATTTTGTTTTAACCAAATCATTTCTTCATGTGTGAGTTTATTGGGATTAAATTCAACACG

CATATTACGTCTATCCCAAGTATCTGCTTTTACTTTGTCATATTCGATATAAACTTTTTCTT

GAAGTGCCTTAGCTTTAAACTTTGTTTGAAGTATATCCCAAAGTCGTATTTGTGGCTCTAC

ACTCATAAAGTCAGATAGCTTTTTAGCATTAGTTTTGTTCAAATTTCCAACGATTGTCATG

GCATCAAAACTTAATGCGGGTTGAGATTTTCCCAAAGTTTGACCACTTAACCGGCTATTA

CTTAACCGGCTATTAGAGACGGAACTAACTCAACGCTAGTAGTGGATTTAATCCCAAATG

AGCCAACAGAACCAGAACCAGAAACAGAACAAGTAACATTGGAGTTAGAAATGGAAGA

AGAAAAAAGCAATGATTTCGTGTGAATAATGCACGAAATCATTGCTTATTTTTTTAAAAA

GCGATATACTAGATATAACGAAACAACGAACTGAATAAAGAATACAAAAAAAGAGCCAC

GACCAGTTAAAGCCTGAGAAACTTTAACTGCGAGCCTTAATTGATTACCACCAATCAATT

AAAGAAGTCGAGACCCAAAATTTGGTAAAGTATTTAATTACTTTATTAATCAGATACTTA

AATATCTGTAAACCCATTATATCGGGTTTTTGAGGGGATTTCAAGTCTTTAAGAAGATACC

AGGCAATCAATTAAGAAAAACTTAGTTGATTGCCTTTTTTGTTGTGATTCAACTTTGATCG

TAGCTTCTAACTAATTAATTTTCGTAAGAAAGGAGAACAGCTGAATGAATATCCCTTTTGT

TGTAGAAACTGTGCTTCATGACGGCTTGTTAAAGTACAAATTTAAAAATAGTAAAATTCG

CTCAATCACTACCAAGCCAGGTAAAAGTAAAGGGGCTATTTTTGCGTATCGCTCAAAAAA

AAGCATGATTGGCGGACGTGGCGTTGTTCTGACTTCCGAAGAAGCGATTCACGAAAATCA

AGATACATTTACGCATTGGACACCAAACGTTTATCGTTATGGTACGTATGCAGACGAAAA

-56CCGTTCATACACTAAAGGACATTCTGAAAACAATTTAAGACAAATCAATACCTTCTTTATT

GATTTTGATATTCACACGGAAAAAGAAACTATTTCAGCAAGCGATATTTTAACAACAGCT

ATTGATTTAGGTTTTATGCCTACGTTAATTATCAAATCTGATAAAGGTTATCAAGCATATT

TTGTTTTAGAAACGCCAGTCTATGTGACTTCAAAATCAGAATTTAAATCTGTCAAAGCAG

CCAAAATAATCTCGCAAAATATCCGAGAATATTTTGGAAAGTCTTTGCCAGTTGATCTAA

CGTGCAATCATTTTGGGATTGCTCGTATACCAAGAACGGACAATGTAGAATTTTTTGATCC

CAATTACCGTTATTCTTTCAAAGAATGGCAAGATTGGTCTTTCAAACAAACAGATAATAA

GGGCTTTACTCGTTCAAGTCTAACGGTTTTAAGCGGTACAGAAGGCAAAAAACAAGTAGA

TGAACCCTGGTTTAATCTCTTATTGCACGAAACGAAATTTTCAGGAGAAAAGGGTTTAGT

AGGGCGCAATAGCGTTATGTTTACCCTCTCTTTAGCCTACTTTAGTTCAGGCTATTCAATC

GAAACGTGCGAATATAATATGTTTGAGTTTAATAATCGATTAGATCAACCCTTAGAAGAA

AAAGAAGTAATCAAAATTGTTAGAAGTGCCTATTCAGAAAACTATCAAGGGGCTAATAG

GGAATACATTACCATTCTTTGCAAAGCTTGGGTATCAAGTGATTTAACCAGTAAAGATTT

ATTTGTCCGTCAAGGGTGGTTTAAATTCAAGAAAAAAAGAAGCGAACGTCAACGTGTTCA

TTTGTCAGAATGGAAAGAAGATTTAATGGCTTATATTAGCGAAAAAAGCGATGTATACAA

GCCTTATTTAGCGACGACCAAAAAAGAGATTAGAGAAGTGCTAGGCATTCCTGAACGGA

CATTAGATAAATTGCTGAAGGTACTGAAGGCGAATCAGGAAATTTTCTTTAAGATTAAAC

CAGGAAGAAATGGTGGCATTCAACTTGCTAGTGTTAAATCATTGTTGCTATCGATCATTA

AATTAAAAAAAGAAGAACGAGAAAGCTATATAAAGGCGCTGACAGCTTCGTTTAATTTA

GAACGTACATTTATTCAAGAAACTCTAAACAAATTGGCAGAACGCCCCAAAACGGACCC

ACAACTCGATTTGTTTAGCTACGATACAGGCTGAAAATAAAACCCGCACTATGCCATTAC

ATTTATATCTATGATACGTGTTTGTTTTTCTTTGCTGTTTAGTGAATGATTAGCAGAAATAT

ACAGAGTAAGATTTTAATTAATTATTAGGGGGAGAAGGAGAGAGTAGCCCGAAAACTTT

TAGTTGGCTTGGACTGAACGAAGTGAGGGAAAGGCTACTAAAACGTCGAGGGGCAGTGA

TATTTGTCCTAATATGATTTTAGCAGTATAATTGACTTGGTGAATAGGTCATTTAAGTTGG

GCATAATAGGAGGAGTAAAATGAAAAAATTTATTTATCGAGTTTTAGAAAATGACGAAG

TGGTGGCTATTTTTAATGAGCAACAATATGCGCAAGATTTTATCGCTTACGAAAAGACAA

TTTCTGATAAGCAATTTGAAATTGAAAAAGTAGATATTGCTGATTGGTTATTGCAACCGA

GAGAATTTTAGAGGTTGGTTGAAAATGGCTAAAATTGGTTATGCACGTGTCAGTAGCAAA

GAACAGAACTTAGATCGGCAATTACAAGCGTTACAGGGCGTTTCTAAGGTCTTTTCAGAC

AAATTAAGCGGTCAATCGGTCGAACGCCCACAATTACAAGCTATGCTTAACTATATTCGT

GAAGGGGATATTGTTATTGTTACTGAATTAGATCGATTAGGACGAAATAATAAAGAATTA

ACAGAATTGATGAATCAAATTCAAATTAAGGGGGCAACCCTGGAAGTCTTAAATTTACCC

TCAATGAATGGTATTGAAGATGAAAATTTAAGGCGTTTGATTAATAGCCTTGTCATTGAA

TTGTACAAGTATCAAGCAGAATCAGAACGAAAAAAAATTAAGGAACGTCAGGCACAAGG

AATCGAAATTGCTAAGAAAAAAGGCAAATTCAAAGGTCGTCAGCATAAATTTAAAGAAA

-57ATGATCCACGTTTAAAGTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGC

TCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAAT

CACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCA

ACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCT

ACGTGCTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACT

ATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGC

ACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATG

ACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCCCCG

GCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGG

ATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAG

CGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTA

CCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACA

CTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGA

CCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTT

ATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAA

AAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCG

ACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTT

GTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATT

GGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACA

TATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTT

GCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAA

ACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCA

GCTCACCGTCTTTCATTGCCATACGG (SEQ ID No: 55). The sequence is exactly aligned with the predicted PSA sequence cloned into pGG55. The LLO-PSA open reading frame is underlined;

lowercase letters indicate the sequence of the PSA itself.

EXAMPLE 8: LISTERIA-LLO-DOG CONSTRUCTIONS ARE ENTITLING CYTOTOXICIC T-LYMPHOXY-SPECIFIC T-LYMPHOCYTES FOR ANTIGENE

BEFORE CANCER-I

EXPERIMENTAL MATERIALS AND METHODS

CTL assays [0259] Male C57BL / 6 mice were immunized with 0.1 LD50 Lm-PSA or 0.1 LD50 LmHPV16E7E6TM ip and a booster was given once every 2 weeks. Spleens were harvested 6 days after boosting. Isolated splenocytes were obtained and stimulated for 5 days with mitomycin treated MC57G infected infected MC57G cells as feed elements. In the first experiment, CTL determination was performed using pulsed PSA H2Db peptide (1 μΜ, HCIRNKSVIL; SEQ ID No: 60) to EL4 cells as targets,

-58 labeled 100 μΜ europium (Sigma), using the following ratios E: T: 25: 1, 8: 1, 2.8: 1, 0.9: 1, 0.3: 1, 0.1: 1 and 0 03: 1. After a 4-hour incubation, targets and effectors were mixed, the cells were separated from the culture supernatant by centrifugation. Release of europium from lysed cells in the supernatant was determined as follows: 10 µl of supernatant was added to 100 µl of europium stimulation solution (Delfia). Absorbance at 590 nm was read using a Victor II spectrophotometer (Perkin Elmer). Maximum europium release was determined from the supernatant of labeled target cells with 1% triton X100, and spontaneous release was determined for target cells incubated in the absence of effector cells. In the second experiment, the E: T ratio was kept constant at 25: 1 and the peptide concentrations were varied as indicated. The percentage of specific lysis was defined as [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100.

Cytokine secretion assays [0260] Male C57BL / 6 mice were immunized with Lm-PSA or Listeria expressing various Wilma tumor antigen fragments (negative control) or were left immunized. Mice were boosted once after two weeks and spleens were harvested 6 days after boost. Isolated splenocytes were obtained and stimulated in vitro overnight in the presence of 1 μΜ of PSA H2D peptide^b. IFN-γ secreted by isolated splenocytes was determined in an ELISpot assay.

Cell Culture, Materials and Reagents [0261] TRAMP-C1 cells of mouse prostate adenocarcinoma obtained from C57BL / 6 mouse prostate cancer were purchased from ATCC. This cell line is negative for PSA expression. Cells were maintained in modified Dulbecco's Eagle medium with 4 mM L-glutamine, adapted to contain 1.5 g / L sodium bicarbonate and 4.5 g / L glucose with the addition of 0.005 mg / mL bovine insulin, 10 nM dehydroisoandrosterone, 90% ; fetal bovine serum, 5% and Nu-Serum IV, 5%. The gene encoding the full-length human PSA protein, including its signal sequence, was subcloned into the pUV6 / v5 plasmid (Invitrogen). After confirming the correct sequence, the plasmid was linearized and transfected into TRAMP-C1 cells using Lipofectamine 2000 ™ (Invitrogen). Positive clones were selected in the presence of 10 pg / ml blasticidin. Several stably expressing PSA clones were isolated and tested for the secretion of human PSA into cell culture medium.

Subcutaneous tumor inoculation [0262] Two different clones of PSA-expressing TRAMP-C1 cells were resuspended after 5 x 10⁶ cells per 200 mcl dose. Male C57BL / 6 mice (8 per group, 6-8 weeks) sc were inoculated into the left flank.

Tumor regression studies [0263] Seven days after tumor inoculation, mice were immunized with 0.1 LD50 Lm-PSA (10⁷ CFU), 0.1 LD50 Lm-HPV16E7 or PBS. Two booster doses were given in

-59 days 15 and 25 after tumor inoculation. Tumors were monitored for 90 days. Tumor size was defined as the average of two perpendicular diameters.

Orthotopic Injection of Prostate Cancer Cells [0264] Six-week-old male C57BL / 6 mice were anesthetized with 2% isoflurane. In the sterile field, an incision was made at the bottom of the midline to gain access to the prostate gland. 1x10 is injected into the left lobe of the dorsal part of the prostate gland⁵ TRAMPC-1 / PSA tumor cells from a single cell suspension in PBS, using a 27G needle attached to a 50-Ham Hamilton Hamilton syringe. Mice were sutured and the sutures were removed 10 days after surgery. Seven days later, mice were immunized iv with Lm-PSA, LmHPV16E7 or PBS. Mice were sacrificed at various time points, the prostate glands were surgically removed and weighed to determine tumor growth.

Tumor protection studies [0265] C57BL / 6 mice were immunized and boosted with Lm-PSA, LmHPV16E7 or PBS as described in the previous example. Seven days after the booster, mice were injected sc 5 x 10⁶ TRAMPC-1 / PSA tumor cells. Tumor growth was monitored by caliper measurement for 90 days.

Inhibition of prostate cancer metastasis [0266] For orthotopic tumor inoculation, 8-10 week old male C57BL / 6 mice (Jackson labs) were anesthetized with isoflurane. An incision was made in the lower abdomen, cranial to the foreskin glands, and seminal vesicles were carefully removed to expose the dorsolateral side of the prostate. 5 x 10 was injected using a 29G insulin needle⁵ TRAMPC-1 / PSA cells suspended in PBS into the dorsolateral prostate in a volume of 20 L. The seminal vesicles and prostate were held for one minute to allow the injected cells to settle in the gland and then gently placed back into the abdominal cavity. The body wall and skin wounds were closed with 5-0 PDS and 5-0 nylon, respectively.

[0267] The tumors were allowed to grow for 50 days. The primary tumor was removed during autopsy and fixed in formalin, then embedded in formalin, cut into sections and stained with H&amp;E. Enlarged lymph nodes from the perineum region were visualized under a surgical microscope and then prepared, fixed, embedded and histologically analyzed for prostate cancer cells.

Tissue Immunostaining [0268] Formalin-fixed prostate tumor tissues are embedded in paraffin, cut into sections, applied to Plus ™ slides (VWR Corp), and then stained using a Dako auto-staining system. Slides are pre-treated with 3.0% hydrogen peroxide for 10 minutes, then washed and treated with 10 Lig / mL Proteinase K solution for 3 minutes to increase antigen recovery. Non-specific binding sites are blocked by adding normal goat serum to

-6030 minutes, and then 10 pg / ml solution of rabbit anti-human PSA antibody (Sigma) or rabbit anti-human Proliferating Cell Nuclear Antigen (AB15497, AbCam antibody) into the tissue for 30 minutes. The primary antibody is removed by washing, and the appropriate horseradish peroxidase-labeled secondary antibody is used for a period of 30 minutes, and detected using NovaRed ™ substrate (Vector Labs, Burlingame, CA) in an 8-minute incubation. Slides are counterstained with hematoxylin before drying.

[0269] Cells from slides with primary sections and lymph nodes are defined as positive or negative for human PSA. Four regions of each slide were randomly selected, and 20 cells from each region were scored. Staining of PSA in tumors is compared to lymph node metastases from the same mouse.

Listeria strains [0270] Listeria vaccines are prepared and stored as described in the previous example.

RESULTS [0271] to test the immunogenicity of LLO-PSA, 6-8 week old C57BL / 6 mice (Jackson Laboratories) were immunized with ip Lm-PSA (0.1 LD50, 1 x 10⁷ CFU per dose) or Lm-HPV16E7E6TM (negative control, 0.1 LD50, 1 x 10⁶ CFU per dose) or left unimmunized. Splenocytes from vaccinated mice were tested for the ability to recognize and lyse PSA peptide presenting cells in vitro in a CTL assay. Splenocytes from immunized mice were able to recognize and lyse pulsed PSA-treated tumor cells with high efficiency (Fig. 20A). In addition, the response was dose-dependent, relative to the amount of antigen presented by the target cells (Fig. 20B).

[0272] In additional assays, mice were immunized with Lm-PSA or strains expressing Wilma tumor antigen fragments (negative control), and cytokine secretion was determined in response to incubation with PSA peptide. Splenocytes from vaccinated mice showed high levels of IFN-γ secretion (Fig. 18).

[0273] Hence, PSA-expressing LM strains and LLO-PSA fusions are effective in the induction of antigen-specific CTLs that are capable of directed cell lysis and IFN-γ secretion. Accordingly, PSA-expressing LM strains and LLO-PSA fusions are effective in the therapeutic and prophylactic vaccination against PSA-expressing prostate cancer.

[0274] The Listeria vaccines described in the previous example are used in cancer protection experiments in an orthotopic prostate cancer animal model. Mice are immunized with Lm-PSA, LmHPV16E7 or PBS, then injected with TRAMPC-1; Lm-PSA protects mice against tumor formation.

[0275] In additional experiments, mice were first injected with TRAMPC-1 / PSA prostate cancer cells, vaccinated with Lm-PSA, LmHPV16E7 or PBS 4

-61 days later, and a booster dose of the same vaccine was given. Lm-PSA hinders the growth of metastases of prostate cancer.

[0276] Hence, PSA-producing LM strains and LLO-PSA fusions induce cancer protection.

EXAMPLE 9: LISTERIA-LLO-DOG CONSTRUCTIONS ARE SPECIAL FOR

TOTOTOXIC ANTIGENIC T lymphocytes PROTECT

BEFORE CANCER-II

EXPERIMENTAL MATERIALS AND METHODS [0277] In vitro macrophage invasion study. Macrophage-like J774A.1 cells were placed 1 x 10⁶ cells per well in 24 well plates. The next day, cells were infected at a MOI of 1: 1 with various Lm strains for 1 hour in the absence of antibiotics. Extracellular bacteria were removed by washing and the cells were incubated in medium containing 50 μg / ml gentamicin, for 30 min at 37 ° C and 5% CO2. Intracellular bacterial growth was monitored by sampling at various time points, up to 8 hours, followed by titration of the bacteria in BHI dishes after lysing the cells with dH2O.

[0278] Antitumor efficacy. TRAMPC-1 is the murine C57BL / 6 murine prostate adenocarcinoma cell line used to construct the PSA secreting prostate tumor model to test the efficacy of Lm-LLO-PSA as a vaccine. The complete open reading frame of human PSA, including its secretory signal sequence at the N-terminus of the molecule was cloned under the control of the UbC promoter in plasmid pUB6 / V5 (Invitrogen). The resulting plasmid (pAdv63) was sequenced to confirm the correctness of the PSA gene sequence and then transfected into the TRAMPC-1 cell line using lipofectamine 2000 (Invitrogen). Transfected cells were selected by growing the cells in medium containing 10 μg / ml blasticidin (Invitrogen). Individual clones were isolated by limiting dilution and screened for PSA secretion into extracellular culture medium using an anti-human PSA ELISA kit. Several positive clones were obtained, propagated and stored in liquid nitrogen. One clone (T-PSA23) was selected for further study.

[0279] Tumor regression study. Three groups of 8 male C57BL / 6 (6-8 week old) sc mice were inoculated into the right flank, 5 x 10⁶ T-PSA23 cells. Seven days later, when tumors reached a mean diameter of 4-5 mm, ip Lm-LLO-PSA (1 x 10 mice) were immunized⁷ CFU per dose), Lm-LLO-E7 (1 x 10⁸ CFU per dose) or left untreated. Lm-LLO-E7 previously described by Gunn et al., Was constructed in the same way as Lm-LLO-PSA, but instead of PSA, it expressed HPV16E7 and was used in this experiment as an unrelated Lm control. Immunizations were repeated twice with 1 week interval. Tumors were monitored weekly for seven weeks. Tail vein blood samples were taken from all experimental animals on day 40 after tumor inoculation and the presence of PSA protein in sera was tested using a PSA ELISA kit. For recorces

Comparative, tumor cells were injected as described above. Mice were immunized twice with Zm-LLO-PSA (1x10⁷ CFU per dose in 200 μΐ PBS), recombinant PSA vaccinia (1 x 10⁷ PFU per dose in 200 μΐ PBS, ip) or a pDNA vaccine containing a mixture of PSA / pCDNA3.1 (100 μg) with GM-CSF / pCDNA3.1 (100 μg) in a volume of 50 μΐ, by intramuscular injection into the left quadriceps muscle. Tumors were monitored once a week using an electronic caliper, and tumor sizes were expressed as the mean of two perpendicular diameters.

Immunogenicity studies in mice [0280] ELISpot assay: Groups of 4-5 male C57BL / 6 mice (6-8 weeks old) were immunized three times with Zm-LLO-PSA or a similar construct containing unrelated antigen as negative control (Zm-LLO-WT1), or not immunized (naive). Six days after the boost, spleens were removed and processed into single cell suspensions. Red blood cells were lysed by incubation with ACK lysis solution for 2 min and then washed twice with C-RPMI medium. Isolated splenocytes were placed 2 x 10 each⁵ cells per well in the presence of 1 µM PSA65-74 peptide (limited to H2D^b PSA HCIRNKSVIL epitope) on ELISpot plates previously coated with the mouse IFN-γ antibody (mAb AN18). Plates were incubated overnight at 37 ° C. Spot forming cells (SFCs) were detected according to the manufacturer's instructions (Mabtech AB). Points were counted using a stereo microscope.

[0281] Intracellular staining for IFN-γ. Splenocytes isolated from immunized, control or naive mice were stimulated at 2x10⁶ cells per well for 5 hours. in 96-well round-bottomed plates, with 1 µM PSA peptide_65-74 or control peptides, in the presence of IL-2 (50 U / ml) and monensin A (StopGolgi, BD Biosciences). Cells incubated in medium without peptide were used as a negative control. After incubation, cells were stained with anti-mouse CD8-FITC, CD3-PerCPCy5.5 and CD62L-APC antibodies. Then, cells were permeabilized and stained with anti-mouse IFNy-PE antibody and analyzed on a FACS Calibur cytometer (BD Biosciences). Data were analyzed using CellQuest Pro software. Cells were gated as CD3 + CD8 + CD62L^low and the number of IFN-Y positive cells was expressed as a percentage of total gated cells.

[0282] Cytotoxicity assay. T-cell cytotoxic activity in response to Zm-LLO-PSA vaccination was measured by a PSA specific CTL assay. Groups of 4 male C57BL / 6 mice were injected with three doses of Zm-LLO-PSA (1x10)⁷CFU) or Zisteria expressing unrelated antigen (HPV16E7E6), one week apart. The naive group did not receive any immunization. Spleens of immunized and naive mice were collected 6 days after the last booster. Splenocytes were incubated for 5 days in C-RPMI medium containing 20 U / ml mouse IL-2 (Sigma) in the presence of PSA / vaccinia infected MC57G cells (MOI 5 for 4 hours) and treated with mitomycin C at an effector: stimulator ratio of 1: twenty. Cytotoxicity assay was performed for 4 hours, incubating effector cells with EL4 target cells, pulsed for 1 hour h2D^b, peptide PSA65-74 and labeled with europium. Europium released

63 cells lysed were measured by mixing a sample of cell culture supernatant with europium stimulating solution (Delfia / Perkin Elmer, Waltham, MA) and absorbance at 590 nm (Perkin Elmer / Wallac, VictorII) was read. Two sets of assays were performed: a) using different effector: target (E: T) ratios at a constant peptide concentration of 1 pM; and b) using different concentrations of PSA peptide at a constant E: T ratio of 25: 1. SC50 values ​​were calculated as the peptide concentration required to obtain 50% of the maximum peptide concentration used (1 pM) minus the background level at 0 pM.

[0283] Analysis of tumor infiltrating lymphocytes (TIL): T-PSA23 cells embedded in matrigel were inoculated sc to the right flank of male C57BL / 6 mice that were immunized on days 7 and 14 with Lm-LLO-PSA, control Lm vaccine or left untreated. On day 20, tumors were surgically excised, washed in ice-cold PBS and minced with a scalpel. The tumor was incubated in matrigel cell recovery solution (BD Biosciences) for 2 hours on ice. TIL was additionally released by mechanically tearing the tissue in a cell strainer using a syringe plunger. Isolated cells were stained with PSA65-74 H-2Db with tetramer-PE and anti-mouse CD8-FITC, CD3-PerCP-Cy5.5 and CD62L-APC antibodies to characterize PSL epitope specific CTLs. To analyze regulatory T cells in the tumor, TIL was also stained with CD4-FITC, CD3-PerCP-Cy5.5 and CD25-APC. Cells were then permeabilized and stained with anti-FoxP3-PE antibody (Milteny Biotec). Cells were analyzed on a FACS Calibur cytometer (BD Biosciences). Data were analyzed using CellQuest Pro software. Regulatory T cells were defined as CD3 cells⁺CD4⁺CD25⁺Fop3⁺.

[0284] Statistical analysis: Non-parametric Mann-Whitney and Kruskal Wallis tests were used to compare tumor sizes between different treatment groups. Tumor sizes were compared on day 40 for statistical analysis because it was the latest time point with the highest number of mice in each group. The tstudent test was used to compare means in ELISpot experiments. A p-value of less than 0.05 was considered statistically significant in these analyzes.

RESULTS [0285] Lm-LLO-PSA is capable of infecting macrophages in vitro. The uptake by antigen presenting cells such as dendritic cells and macrophages is an essential requirement for Lm-based vaccines for the successful delivery and presentation of antigens to the immune system. This property can be tested in vitro by infecting the mouse macrophage-like J774A.1 cell line and quantifying intracellular bacterial growth, which is an indication that the Lm vaccine strain was able to properly enter cells and multiply. This assay showed that Lm-LLO-PSA was able to invade macrophages and grow using a similar solution to the Lm wild-type strain 10403s. In contrast, the Lm XFL7 strain, which lacks prfA and is not able to escape from the phagolysosome, is not able to proliferate in J774A.1 cells and its titre

Remains constant several hours after infection (Fig. 14). PrfA complementation in the Lm XFL7 strain with the plasmid pAdv34 results in restoration of the invasive in vitro properties of the strain, to a degree comparable to that of the wild-type strain.

[0286] Lm-LLO-PSA causes regression of pre-established PSA-expressing tumors in mice. The efficacy of Lm-LLO-PSA in causing regression of PSA-expressing tumors was tested in a mouse model, using a PSA transfected TRAMPC-1 cell line. TrampC-1 cells, originally derived from a murine tumor - prostate adenocarcinoma - was stably transfected with the human PSA gene under the control of the human ubiquitin C promoter. Secretion of PSA by transfected cells was confirmed by ELISA. Four clones were propagated and passaged for 25 generations in the absence of the antibiotic selection marker blasticidin. Cell culture supernatants were tested for PSA secretion levels, which were found to be comparable between cells at early passages and passage 25 (data not shown). One of these clones retained carcinogenicity in vivo and formed solid tumors after inoculation with sc in male C57BL / 6 mice. An early passage of this clone was used in cancer research and is referred to as T-PSA23. Three groups of 8 mice were inoculated sc 5 x 10⁶ T-PSA23 cells and monitored daily for tumor growth. After 7 days, a solid tumor mass of 4-5 mm in diameter was detected in 7 out of 8 mice in each group (Fig. 15). Then the mice were immunized with Lm-LLO-PSA (1x10⁷ CFU / mouse), Lm-LLOE7 (1x 10⁸ CFU / mouse) or left untreated. The primary immunization was followed by two booster doses, 1 week apart, with the same dose as the original. The mice (7 out of 8) in each control group developed slow-growing tumors that reached a diameter of 2 cm within 7 weeks after inoculation (Fig. 15), at which point the mice were sacrificed. One mouse in each group showed no signs of cancer at any time. Five days after the first immunization, a reduction in tumor size was observed in 7 of 8 mice in the Lm-LLO-PSA immunized group, and complete tumor regression was observed in 5 of these mice after 1 week. One of the tumors in the Lm-LLO-PSA immunized group remained stable until the third immunization. However, one week after the third vaccination, this tumor also regressed and became undetectable. Another mouse in this group developed a slowly growing tumor that reached 18-20 mm in 7 weeks, and the mouse was sacrificed. At the end of the study, at week 8 after tumor inoculation, 7 out of 8 mice in the Lm-LLO-PSA immunized group were still tumor free. This experiment was repeated twice with similar results. Nonparametric statistical analysis - Kruskal-Wallis tests - was performed on tumor size on day 40, before killing naive and Zm-LLO-E7 mice. There were no statistically significant differences between tumor sizes of naive mice and LmLLO-E7 immunized mice (p = 0.613). However, Lm-LLO-PSA immunization has been shown to have a significant effect on T-PSA23 tumor growth (p = 0.002). Serum samples were taken from controls and Lm-LLO-PSA immunized mice on day 40 after tumor challenge and tested for the presence of PSA using the anti-human PSA ELISA kit (American Qualex). Significant amounts of PSA (5-10 ng / ml) were detected in the serum of control mice and in mice in the Lm-65LLO-PSA group that had tumors. No PSA was found in the serum of Lm-LLO-PSA immunized mice after tumor regression.

[0287] Lm-LLO-PSA elicits cellular immune responses against PSA in both spleens and tumors. To clarify whether vaccination with Lm-LLO-PSA induced PSA-specific CTL capable of infiltrating the tumor, mice were implanted sc with a mixture of T-PSA23 + matrigel cells and immunized with two doses of Lm-LLOPSA, Lm-LLO-E7 or none. Spleens and tumors were harvested 6 days after the last immunization and tested for the presence of a CD8 positive T cell tetramer⁺/ PSA65-74 (with H2D limitation^b) using FACS analysis. No significant antiPSA responses were observed in the spleens of naive mice (0.05%) or Lm-LLO-E7 vaccinated mice (0.09%) (Fig. 16, upper panel). However, after immunization with Lm-LLO-PSA, a significant number of PSA specific CD8 T cells⁺ found in spleens. Interestingly, when tumors excised from these mice were tested for TIL, low numbers of PSA-specific CD8 + T cells were identified in both naive and Lm-LLO-E7 immunized mice (1.5% and 0.51%, respectively). Immunization with Lm-LLO-PSA resulted in a significant increase in this number, as 16.1% of all CD8⁺ TIL appeared to be specific for PSA65-74 (Fig. 16 bottom panel).

[0288] Immunization of Lm-LLO-PSA causes a decrease in regulatory T cells in tumors, but not in spleens. Presence of CD4 regulatory T cells⁺/ CD25⁺/ FoxP3⁺ (Treg) cancer infiltrators have been shown to be associated with an increased risk of cancer progression. Consequently, we examined the presence of this T cell population in tumors of Lm-LLO-PSA immunized mice or control mice. Interestingly, immunization with Lm-LLO-PSA or Lm-LLO-E7 caused a significant decrease in the incidence of Tregs in tumors compared to tumors in naive mice (Fig. 17). In fact, Lm-LLO-PSA had a slightly greater effect than Lm-LLO-E7 on the frequency of cancer infiltrating Tregs.

On the other hand, no significant differences were found between the percentages of CD4 cells⁺/ CD25⁺/ FoxP3⁺T isolated from the spleen of Lm-LLO-PSA mice or control mice.

[0289] Immunization with Lm-LLO-PSA results in strong cellular immune responses against PSA. It has been shown that immune responses induced by Lm-based vaccines result in cellular rather than humoral responses. Activation and functionality of CD8 T cells was studied⁺ induced by Lm-LLO-PSA in the spleens of immunized mice.

[0290] Cytokine secretion. IFN-γ secretion by activated T cells in response to in vitro stimulation with H2D peptide^b PSA was tested by ELISpot and intracellular cytokine staining. Mice were immunized three times with Lm-LLOPSA or Lm-LLO-WT-1 (Lm-LLO-Wilma tumor antigen as a negative control) or not immunized (naive). Isolated splenocytes were then prepared and incubated with PSA peptide and IFN-γ secreting CD8 + cells were quantified. In both assays, Lm-LLO-PSA immunized mice showed a significantly higher number of IFN-γ secreting cells in response to pulsed peptide treatment than

-66 negative controls (p &lt;0.001). The number of IFN-γ secreting cells measured in the ELISpot assay was approximately 11-fold higher than in controls (Fig. 18). When testing for intracellular cytokine staining for IFN-γ, a 30-fold increase in the number of CD8 + CD62L cells was detected^low secreting IFN-γ from mice immunized with ZmLLO-PSA compared to control (4.22% for Zm-LLO-PSA vs. 0.15% for naive) (Fig. 19).

[0291] Cytotoxicity assay (CTL). To determine the functional activity of PSA-specific T cells, splenocytes from immunized or naive mice were tested in a CTL assay against cells treated with pulsed h2D peptide^bPSA. Using a constant peptide concentration (1 µM), splenocytes from Zm-LLO-PSA immunized mice showed a maximum specific lysis of 60%, which decreased in proportion to the E: T ratio (Fig. 20A). This result depended on the peptide concentration in the lysis assay, with the maximum value achieved at a peptide concentration of only 0.1 µM (Fig. 20B). SC value₅₀ The polyclonal population, which is a useful measure of average T cell avidity, was approximately 1 pM. The results from this experiment also confirm that the immunization with Zm-LLO-PSA generated specific high avidity cytolytic T cells, capable of lysing cells that display the PSA target antigen.

[0292] Comparison of the anti-tumor activity of Lm-LLO-PSA with other PSA-based vaccines. Finally, to further evaluate the Zm-LLO-PSA vaccine, its antitumor efficacy was compared with two other PSA-based vaccines that have been described in the literature, i.e. recombinant DNA and vaccinia-based vaccine. To this end, the complete PSA open reading frame for pCDNA3.1 was cloned under the control of the hCMV promoter (pAdv40.1). A similar DNA vaccine has previously been described in Roos et al. using a similar vector backbone, i.e. pVax (Invitrogen). The difference between the two vector skeletons (pCDNA3.1 and pVax) was only in their antibiotic marker for selection. pAdv40.1 was injected as a mixture with GMCSF / pCDNA, which was used as an effective adjuvant in other DNA vaccines. Expression of PSA from the PSA / pCDNA plasmid was confirmed by in vitro transfection into the 293FT cell line (data not shown). Recombinant PSA-vaccinia was previously described in Hudge et al. Mice were implanted with tumor T-PSA23 cells as described above and then immunized twice with each vaccine, the optimal dose and route of administration as reported in the literature. Immunization with PSA / vaccinia had no effect on the growth of T-PSA23 tumors as all naive and immunized with PSA / vaccinia mice developed tumors that reached 20 cm in 7-8 weeks, and the mice had to be killed (Fig. 21b ). However, 1 in 8 mice immunized with the pSA / DNA vaccine and 3 in 8 mice vaccinated with Zm-LLO-PSA remained tumor free for 90 days, at which time the study was terminated (Fig. 21A and insert in the table). This experiment was repeated twice with similar results. Hence Zm-LLO-PSA proved to be the most effective vaccine of the three types of PSA-based vaccines for regression purposes established PSA-expressing tumors. It should be noted that in comparative studies, due to the limited availability of the other two vaccines, only two doses were administered. Consequently, Zm-LLO-PSA proved to be less effective (complete regression)

Cancer was observed in only 3 out of 8) than when injected three times (when 7 out of 8 tumors regressed, as seen in Fig. 15). This observation was confirmed in several other studies (data not shown).

COMPARATIVE EXAMPLE 10: LISTERIA-LLO-HYDROLAASE CONSTRUCTIONS

FOLIANOWA 1 (FOLH1) AND LISTERIA-LLO-ANTIGENIC CELLS

OF STEM STEPULAR GRAND (PSCA) ENERGY SPECIAL

CYTOTOXIC LYMPHOCYTE ANTIGEN

EXPERIMENTAL MATERIALS AND METHODS

Growth and storage of bacterial vaccine strains [0293] Recombinant Listeria-LLO-FOLH1 and Listeria-LLO-PSCA were cultured and maintained as described for Lister-in-PSA in Example 7 above.

RESULTS [0294] The gene encoding the truncated FOLH1, which contains the complete FOLH1 open reading frame, except for its secretory signal sequence, is coupled to the gene encoding the truncated nonhemolytic fragment of Listeriolysin O using a similar solution to that described for KLK3 in Example 7 above . Similarly, the gene encoding the truncated PSCA, which contains the complete PSCA open reading frame, except for its secretory signal sequence, is coupled to the gene encoding the truncated nonhemolytic fragment of Listeriolysin O. Each gene is cloned into the Listeria plasmid pGG55 and electropolated into LM XFL-7. The LLO-FOLH1 protein and the LLO-PSCA protein are consequently expressed and secreted episomally from separate recombinant Listeria strains.

[0295] To test the immunogenicity of LLO-FOLH1 and LLO-PSCA, mice were re-immunized with Lm-LLO-FOLH1, Lm-LLO-PSCA or LmWT1A (control - unrelated antigen) or PBS (negative control) as described for LLO-KLK3 in Example 7 above. After culturing with stimulating cells infected with vaccinia-PSA for 5 days, splenocytes from FOLH1 vaccinated mice are capable of recognizing and lysing tumor cells pulsed with FOLH1 peptide, with high efficiency in CTL assay, and splenocytes from PSCA vaccinated mice are capable of recognizing and lysing tumor cells pulsed with PSCA peptide with high efficiency in a CTL assay. In addition, splenocytes exhibit high levels of IFN-γ secretion in response to incubation with FOLH1 peptide or PSCA peptide, respectively.

[0296] Hence, FOLH1-expressing LM strains, expressing PSCA LM strains, LLO-PSCA fusions and LLO-FOLH1 fusions are effective in the induction of antigen-specific CTLs that are capable of directed cell lysis and IFN-γ secretion. Accordingly, FOLH1-expressing LM strains, expressing PSCA LM strains, LLO-PSCA fusions and LLO-FOLH1 fusions are effective in the therapeutic and prophylactic vaccination against PSA-expressing prostate cancer.

-68 COMPARATIVE EXAMPLE 11: CONSTRUCTIONS LISTERIA-LLO-FOLH1

PROTECT AGAINST CANCER [0297] The Listeria vaccines described in the previous Example are used in cancer protection experiments in the orthotopic prostate cancer animal model described in Examples 8 and 9 above. Mice are immunized with LmFOLH1, Lm-PSCA, LmWT1A or PBS and then injected into PC3M-LN4 or 22Rv1 cells. Lm-FOLH1 and Lm-PSCA protect mice against tumor formation.

[0298] In additional experiments, mice were first injected with PC-3M prostate cancer cells as described in Examples 8 and 9 above, vaccinated with Lm-FOLH1, Lm-PSCA, LmWT1A or PBS 4 days later, and administered with the same vaccine . Lm-FOLH1 and Lm-PSCA inhibit the growth of metastases of prostate cancer.

[0299] Hence, FOLH1- and PSCA producing LM strains and Lm-FOLH1 and Lm-PSCA fusions induce cancer protection.

LIST OF SEQUENCES [0300] &lt;110&gt; Members of the Board of the University of Pennsylvania ADVAXIS &lt;120&gt; COMPOSITIONS AND METHODS CONTAINING KLK3, PSCA OR FOLH1 Antigens &lt;130&gt; P-7769-PC2 &lt;150&gt; 11 / 798,177 &lt;151&gt; 2007-05-10 &lt; 160&gt; 64 &lt;170&gt; PatentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 1

-69Lys Glu Asn Cheese ile Cheese Cheese Met Ala Pro Pro Ala Cheese Pro Pro Ala 15 10 15

Ser Pro Lys Thr Pro ile Glu Lys Lys His Ala Asp Glu ile Asp Lys 20 25 30 &lt;210&gt; 2 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 2

Lys Thr Glu Glu Gin Pro Cheese Glu val Asn Thr Gly Pro Arg 15 10 &lt;210&gt; 3 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 3

<td>lys</td><td>ala</td><td>Cheese</td><td>val</td><td>Thr</td><td rowspan="2">ASP</td><td>Thr</td><td>Glu cheese</td><td>Gly</td><td rowspan="2">Asp</td><td rowspan="2">Leu Asp cheese</td><td>Cheese Met</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td> 10</td><td> 15</td>

<td>Gin</td><td>Cheese</td><td>ala</td><td>Asp</td><td>Glu</td><td>Cheese</td><td>Thr</td><td>pro Gin</td><td>pro</td><td>Leu</td><td>lys</td><td></td>

25 &lt;210&gt; 4 &lt;211&gt; 20 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 4

Lys Asn Glu Glu val Asn Ala Ser Asp Phe Pro Pro Pro Pro Thr Asp 15 10 15

Glu Glu Leu Arg 20 &lt;210&gt; 5 &lt;211&gt; 33 &lt;212&gt; PRT

-70 &lt;213&gt; Listeria monocytogenes &lt;400&gt; 5

Arg Gly Gly Ile pro Thr ser Glu Glu Phe Cheese Leu Asn Cheese Gly 15 10 15

Asp Phe Thr Asp Asp Glu Asn ser Glu Thr Thr Glu Glu Glu Ile Asp 20 25 30

Arg &lt;210&gt; 6 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Streptococcus pyogenes &lt;400&gt; 6

Lys Gin Asn Thr Ala Ser Thr Glu Thr Thr Thr Thr Asn Glu Gin Pro 1.5 10 15

Lys &lt;210&gt; 7 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Streptococcus equisimilis &lt;400&gt; 7

Lys Gin Asn Thr Ala Asn Thr Glu Thr Thr Thr Thr Asn Glu Gin Pro 15 10 15

Lys &lt;210&gt; 8 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

-71 &lt;223&gt; chemically synthesized &lt;400&gt; 8 ggctcgagca tggagataca cc 22 &lt;210&gt; 9 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; chemically synthesized &lt;400&gt; 9 ggggactagt ttatggtttc tgagaaca 28 &lt;210&gt; 10 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; chemically synthesized &lt;400&gt; 10 gggggctagc cctcctttga ttagtatatt c 31 &lt;210&gt; 11 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial

-72&lt;220&gt;

&lt;223&gt; chemically synthesized &lt;400&gt; 11 ctccctcgag atcataattt acttcatc 28 &lt;210&gt; 12 &lt;211&gt; 55 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; chemically synthesized &lt;400&gt; 12 gactacaagg acgatgaccg acaagtgata acccgggatc taaataaatc cgttt &lt;210&gt; 13 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; chemically synthesized &lt;400&gt; 13 cccgtcgacc agctcttctt ggtgaag 27 &lt;210&gt; 14 &lt;211&gt; 100 &lt;212&gt; PRT

-73 &lt;213&gt; Listeria monocytogenes &lt;400&gt; 14

Met Arg Ala Met Met val val Phe Ile Thr Ala Asn cys Ile Thr Ile 15 10 15

Asn Pro Asp how much Phe Ala Ala Thr Asp Ser Glu Asp Ser Ser Leu 20 25 30

Asn Thr Asp Glu Trp Glu Glu Glu Lys Thr Glu Glu Gin Pro Ser Glu 35 40 45 val Asn Thr Gly Pro Arg Tyr Glu Thr Ala Arg Glu Val Cheese Ser Arg 50 55 60

<td>ASp</td><td>how much</td><td>lys</td><td>Glu</td><td>Leu</td><td>Glu</td><td>lys</td><td>Cheese</td><td>own</td><td>lys</td><td>val</td><td>Arg</td><td>own</td><td>Thr</td><td>own</td><td>lys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>ala</td><td>Asp</td><td>Leu</td><td>how much</td><td>ala</td><td>Underworld</td><td>Leu</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>Glu</td><td>lys</td><td>Gly</td><td>Pro</td><td>own</td>

90 95 how much Asn Asn Asn

100 5 &lt;210&gt; 15 &lt;211&gt; 390 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 15

<td>Underworld 1</td><td>Arg</td><td>ala</td><td>Underworld</td><td>Underworld 5</td><td>val</td><td>val</td><td>phe</td>

<td>own</td><td>Pro</td><td>Asp</td><td>How much twenty</td><td>How much</td><td>phe</td><td>ala</td><td>ala</td>

<td>own</td><td>Thr</td><td>Asp 35</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Glu 40</td>

<td>val</td><td>own 50</td><td>Thr</td><td>Gly</td><td>pro</td><td>Arg</td><td>Tyr 55</td><td>Glu</td>

<td>Asp 65</td><td>How much</td><td>lys</td><td>Glu</td><td>Leu</td><td>Glu 70</td><td>lys</td><td>Cheese</td>

<td>ala</td><td>Asp</td><td>Leu</td><td>how much</td><td>ala 85</td><td>Underworld</td><td>Leu</td><td>lys</td>

<td>How much</td><td>own</td><td>own</td><td>own 100</td><td>own</td><td>Cheese</td><td>glu</td><td>gin</td>

<td>glu</td><td>Ala</td><td>will 115</td><td>Gly</td><td>Ala</td><td>asp</td><td>Arg</td><td>pro 120</td>

<td>pro</td><td>Gly 130</td><td>Leu</td><td>pro</td><td>will</td><td>asp</td><td>will 135</td><td>Ala</td>

<td>Ala</td><td>ile</td><td>Ala</td><td>will</td><td>will</td><td>asp</td><td>will</td><td>glu</td>

Thr Ile Thr Ala ASN CYS ile ile 10 15

Thr Glu Asp Asp will Leu will be 25 30

Glu Glu Lys Thr Glu will Gin Pro 45

Glu Thr Arg Ala Val Arg will be 60

Lys Arg val asn asn asn Lys Thr 75 80

Lys Glu Ala Glu Gly Lys Pro ASN 90 95

ASN Glu Thr Ala Glu Ala ile NSA 105 110

Ile val Ala Glu Arg Gin Arg His 125

Ala Glu Lys Lys Arg Ile Lys 140 Arg

Thr Tyr Leu Leu Glu will Asp Pro

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td>II \ J ^</td><td>· 1 V *.</td><td>-'- d</td><td> 155</td><td> *·..»</td><td></td><td></td><td></td><td> 160</td>

<td>Lys</td><td>pro</td><td>thr</td><td>Lys</td><td>Val</td><td>asn</td><td>Lys</td><td>Lys</td><td rowspan="2">Lys</td><td>val</td><td>Ala</td><td>Lys</td><td>glu</td><td>will</td><td>val</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asp</td><td>Ala</td><td>will</td><td>glu</td><td>will</td><td rowspan="2">asp</td><td>Leu</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Met</td><td>gin</td><td>will</td><td>Ala</td><td rowspan="2">asp</td><td>glu</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td>

<td>will</td><td>will</td><td>pro</td><td>gin</td><td>pro</td><td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>asn</td><td>gin</td><td>gin</td><td>pro</td><td>Phe</td><td>Phe</td><td>pro</td><td rowspan="2">Lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>val</td><td>Phe</td><td>Lys</td><td>Lys</td><td>ile</td><td rowspan="2">Lys</td><td>asp</td><td>for a</td><td rowspan="2">Gly</td><td>Lys</td><td>trp</td><td>Val</td><td>Arg</td><td>asp</td><td>Lys</td><td>ile</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>asp</td><td>glu</td><td>asn</td><td>pro</td><td>glu</td><td>val</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Ala</td><td>Ile</td><td>val</td><td rowspan="2">asp</td><td>Lys</td><td>will</td><td>Ala</td><td>Gly</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>Leu</td><td>Ile</td><td rowspan="2">asp</td><td>gin</td><td>Leu</td><td>Leu</td><td>thr</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Lys</td><td>will</td><td>glu</td><td>glu</td><td>val</td><td>asn</td><td>Ala</td>

<td></td><td></td><td></td><td> 245</td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>will</td><td rowspan="2">asp</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>asp</td><td>glu</td><td>glu</td><td>Leu</td><td rowspan="2">Arg</td><td>Leu</td><td>Ala</td><td>Leu</td>

<td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>pro</td><td>glu</td><td>thr</td><td>pro</td><td>Met</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Phe</td><td>asn</td><td>Ala</td><td>pro</td><td>Ala</td><td>thr</td><td>will</td><td>glu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>will</td><td>will</td><td>Phe</td><td>glu</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>asp</td><td>glu</td><td>glu</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td>thr</td><td>pro</td><td>Met</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Phe</td><td>asn</td><td>Ala</td><td>pro</td><td>Ala</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>thr</td><td>will</td><td>glu</td><td>pro</td><td>will</td><td>will</td><td>Phe</td><td>glu</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>glu</td><td rowspan="2">asp</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td>

<td>glu</td><td>Leu</td><td>glu</td><td>ile</td><td>Ile</td><td rowspan="2">Arg</td><td>glu</td><td>thr</td><td>Ala</td><td>will</td><td>will</td><td>Leu</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>thr</td><td>Arg</td><td>Gly</td><td>asp</td><td>Leu</td><td>Ala</td><td>will</td><td>Leu</td><td rowspan="2">Arg</td><td>asn</td><td>Ala</td><td>ile</td><td>asn</td><td rowspan="2">Arg</td><td>It's</td><td>will</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td> 365</td><td></td><td></td>

<td>gin</td><td>asn</td><td>Phe</td><td>will</td><td rowspan="2">asp</td><td>Phe</td><td>pro</td><td>pro</td><td>ile</td><td>pro</td><td>thr</td><td>glu</td><td>glu</td><td>glu</td><td>Leu</td><td>asn</td>

<td></td><td> 370</td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Arg</td><td>Gly</td><td>Gly</td><td>Arg</td><td>pro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 16 &lt;211&gt; 1170 &lt;212&gt; DNA

-76 &lt;213&gt; Listeria monocytogenes &lt;400&gt; 16

<td>atgcgtgcga</td><td>tgatggtggt</td><td>tttcattact</td><td>gccaattgca</td><td>ttacgattaa</td><td>ccccgacata</td><td> 60</td>

<td>atatttgcag</td><td>cgacagatag</td><td>cgaagattct</td><td>agtctaaaca</td><td>cagatgaatg</td><td>ggaagaagaa</td><td> 120</td>

<td>aaaacagaag</td><td>agcaaccaag</td><td>cgaggtaaat</td><td>acgggaccaa</td><td>gatacgaaac</td><td>tgcacgtgaa</td><td> 180</td>

<td>gtaagttcac</td><td>gtgatattaa</td><td>agaactagaa</td><td>aaatcgaata</td><td>aagtgagaaa</td><td>tacgaacaaa</td><td> 240</td>

<td>gcagacctaa</td><td>tagcaatgtt</td><td>gaaagaaaaa</td><td>gcagaaaaag</td><td>gtccaaatat</td><td>caataataac</td><td> 300</td>

<td>aacagtgaac</td><td>aaactgagaa</td><td>tgcggctata</td><td>aatgaagagg</td><td>cttcaggagc</td><td>cgaccgacca</td><td> 360</td>

<td>gctatacaag</td><td>tggagcgtcg</td><td>tcatccagga</td><td>ttgccatcgg</td><td>atagcgcagc</td><td>ggaaattaaa</td><td> 420</td>

<td>aaaagaagga</td><td>aagccatagc</td><td>atcatcggat</td><td>agtgagcttg</td><td>aaagccttac</td><td>ttatccggat</td><td> 480</td>

<td>aaaccaacaa</td><td>aagtaaataa</td><td>gaaaaaagtg</td><td>gcgaaagagt</td><td>cagttgcgga</td><td>tgcttctgaa</td><td> 540</td>

<td>agtgacttag</td><td>attctagcat</td><td>gcagtcagca</td><td>gatgagtctt</td><td>caccacaacc</td><td>tttaaaagca</td><td> 600</td>

<td>aaccaacaac</td><td>catttttccc</td><td>taaagtattt</td><td>aaaaaaataa</td><td>aagatgcggg</td><td>gaaatgggta</td><td> 660</td>

<td>cgtgataaaa</td><td>tcgacgaaaa</td><td>tcctgaagta</td><td>aagaaagcga</td><td>ttgttgataa</td><td>aagtgcaggg</td><td> 720</td>

<td>ttaattgacc</td><td>aattattaac</td><td>caaaaagaaa</td><td>agtgaagagg</td><td>taaatgcttc</td><td>ggacttcccg</td><td> 780</td>

<td>ccaccaccta</td><td>cggatgaaga</td><td>gttaagactt</td><td>gctttgccag</td><td>agacaccaat</td><td>gcttcttggt</td><td> 840</td>

<td>tttaatgctc</td><td>ctgctacatc</td><td>agaaccgagc</td><td>tcattcgaat</td><td>ttccaccacc</td><td>acctacggat</td><td> 900</td>

<td>gaagagttaa</td><td>gacttgcttt</td><td>gccagagacg</td><td>ccaatgcttc</td><td>ttggttttaa</td><td>tgctcctgct</td><td> 960</td>

<td>acatcggaac</td><td>cgagctcgtt</td><td>cgaatttcca</td><td>ccgcctccaa</td><td>cagaagatga</td><td>actagaaatc</td><td> 1020</td>

<td>atccgggaaa</td><td>cagcatcctc</td><td>gctagattct</td><td>agttttacaa</td><td>gaggggattt</td><td>agctagtttg</td><td> 1080</td>

<td>agaaatgcta</td><td>ttaatcgcca</td><td>tagtcaaaat</td><td>ttctctgatt</td><td>tcccaccaat</td><td>cccaacagaa</td><td> 1140</td>

<td>gaagagttga</td><td>acgggagagg</td><td>cggtagacca</td><td></td><td></td><td></td><td> 1170</td>

&lt;210&gt; 17 &lt;211&gt; 529 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 17

-77Met Lys Lys Ile Met Leu Leu val ile ile Phe Thr Leu Leu will val 15 10 15

Pro ile Ala Gin Gin Ala Glu Lys Thr Asp Ala Ala Phe will ASN Lys 20 25 30

Glu Ile ASN will be going Met Ala Pro Pro Pro Pro Ala Ala Will Will 35 40 45

Pro Lys Thr Lys Pro ile Lys Glu Asp Ala His Lys Glu Asp Ile Tyr 50 55 60

Gin Asp Ile Leu Gly Tyr Lys NSA NSA NSA val val Tyr Leu Gly His 65 70 75 80

<td rowspan="2">asp</td><td colspan="2" rowspan="2">Ala val</td><td colspan="2" rowspan="2">Thr ASN 85</td><td rowspan="2">val</td><td colspan="9">Pro Pro Gly Lys Arg Gly Tyr Lys Asp</td><td rowspan="2">asn</td>

<td colspan="6"> 90</td><td colspan="3"> 95</td>

<td>glu</td><td rowspan="2">tyr</td><td>ile</td><td>val</td><td>val</td><td>glu</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Lys</td><td rowspan="2">Lys</td><td>will</td><td>ile</td><td>asn</td><td>gin</td><td>asn</td><td>asn</td>

<td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ala</td><td rowspan="2">asp</td><td>ile</td><td>gin</td><td>val</td><td>val</td><td>asn</td><td>Ala</td><td>ile</td><td>will</td><td>will</td><td>Leu</td><td>thr</td><td rowspan="2">tyr</td><td>pro</td><td rowspan="2">Gly</td>

<td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td>

<td>Ala</td><td>Leu</td><td>val</td><td rowspan="2">Lys</td><td>Ala</td><td>asn</td><td>will</td><td>glu</td><td>Leu</td><td>Val</td><td>glu</td><td>asn</td><td>gin</td><td>pro</td><td rowspan="2">asp</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>

<td>Leu</td><td>pro</td><td>val</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Ile</td><td rowspan="2">asp</td><td>Leu</td><td>pro</td><td>Gly</td>

<td> 145</td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Met</td><td>thr</td><td>asn</td><td>gin</td><td>asp</td><td>asn</td><td rowspan="2">Lys</td><td>ile</td><td>val</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>Ala</td><td>thr</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asn</td><td>val</td><td>asn</td><td>asn</td><td>Ala</td><td>Val</td><td>asn</td><td>thr</td><td>Leu</td><td>Val</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>asn</td><td>glu</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>tyr</td><td>Ala</td><td>gin</td><td>Ala</td><td rowspan="2">tyr</td><td>pro</td><td>asn</td><td>val</td><td>will</td><td>Ala</td><td rowspan="2">Lys</td><td>ile</td><td>asp</td><td rowspan="2">tyr</td><td>asp</td><td rowspan="2">asp</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td>

<td>glu</td><td>Met</td><td>Ala</td><td rowspan="2">tyr</td><td>will</td><td>glu</td><td>will</td><td>gin</td><td>Leu</td><td>Ile</td><td>Ala</td><td>Lys</td><td>Phe</td><td rowspan="2">Gly</td><td>thr</td><td>Ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>Val</td><td>asn</td><td>asn</td><td>will</td><td>Leu</td><td>asn</td><td>val</td><td>asn</td><td>Phe</td><td rowspan="2">Gly</td><td>Ala</td><td>Ile</td><td>will</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>glu</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td><td>Met</td><td>n gl</td><td>glu</td><td>glu</td><td>val</td><td>ile</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>gin</td><td>ile</td><td>tyr</td><td rowspan="2">tyr</td>

<td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td>

<td>asn</td><td>val</td><td>asn</td><td>val</td><td>asn</td><td>glu</td><td>pro</td><td>thr</td><td>Arg</td><td>pro</td><td>will</td><td rowspan="2">Arg</td><td>Phe</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>Ala</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td><td>gin</td><td>Leu</td><td>gin</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>Val</td><td>asn</td><td>Ala</td><td>glu</td><td>asn</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>pro</td><td>Ala</td><td>tyr</td><td>Ile</td><td>will</td><td>will</td><td>val</td><td>Ala</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Arg</td><td>n gl</td><td>val</td><td rowspan="2">tyr</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>will</td><td>thr</td><td>asn</td><td>will</td><td>His</td><td>will</td><td>thr</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Ala</td><td>Ala</td><td>Phe</td><td>asp</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td> 320</td>

<td>Ala</td><td>Ala</td><td>Val</td><td>will</td><td>Gly</td><td rowspan="2">Lys</td><td>will</td><td>val</td><td>will</td><td>Gly</td><td rowspan="2">ASP</td><td>val</td><td>glu</td><td>Leu</td><td>thr</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>ile</td><td>Ile</td><td>Lys</td><td>asn</td><td>will</td><td>will</td><td>Phe</td><td>Lys</td><td>Ala</td><td>val</td><td>Ile</td><td>tyr</td><td>Gly</td><td>Gly</td><td>will</td><td>Ala</td>

340 345 350

<td>Lys</td><td>ASP</td><td>glu</td><td>val</td><td>gin</td><td>Ile</td><td>Ile</td><td>asp</td><td rowspan="2">Gly</td><td>asn</td><td>Leu</td><td rowspan="2">Gly</td><td>asp</td><td>Leu</td><td>Arg</td><td>asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>ile</td><td>Leu</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td rowspan="2">Gly</td><td>Ala</td><td>thr</td><td>Phe</td><td>asn</td><td rowspan="2">Arg</td><td>glu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td>val</td><td>pro</td>

<td></td><td> 370</td><td></td><td> 375</td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td>

<td>ile</td><td>Ala</td><td rowspan="2">tyr</td><td>thr</td><td>thr</td><td>asn</td><td>Phe</td><td>Leu</td><td rowspan="2">Lys</td><td rowspan="2">asp</td><td>asn</td><td>glu</td><td>Leu</td><td>Ala</td><td>Val</td><td>ile</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Lys</td><td>asn</td><td>asn</td><td>will</td><td>glu</td><td>tyr</td><td>ile</td><td>glu</td><td>thr</td><td>thr</td><td>will</td><td>Lys</td><td>Ala</td><td>tyr</td><td>thr</td><td>asp</td>

405 410 415

<td>Gly</td><td>Lys</td><td>ile</td><td>asn</td><td>Ile</td><td>asp</td><td>It's</td><td>will</td><td>Gly</td><td>Gly</td><td>tyr</td><td>val</td><td>Ala</td><td>gin</td><td>Phe</td><td>asn</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>ile</td><td>will</td><td>trp</td><td>asp</td><td>glu</td><td>val</td><td>asn</td><td>tyr</td><td>asp</td><td>pro</td><td>glu</td><td>Gly</td><td>asn</td><td>glu</td><td>Ile</td><td>val</td>

435 440 445

<td>gin</td><td>His</td><td>Lys</td><td>asn</td><td>trp</td><td>will</td><td>glu</td><td>asn</td><td>asn</td><td>Lys</td><td>will</td><td>Lys</td><td>Leu</td><td>Ala</td><td>It's</td><td>Phe</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>thr</td><td>will</td><td>will</td><td>ile</td><td>tyr</td><td>Leu</td><td>pro</td><td>Gly</td><td>asn</td><td>Ala</td><td>Arg</td><td>asn</td><td>Ile</td><td>asn</td><td>val</td><td>tyr</td>

465 470 475 480

Ala Glu Lys Thr Gly Leu Ala CYS TRP TRP TRP Glu Thr Arg val ile 485 490 495

<td>asp</td><td>asp</td><td>Arg</td><td>asn 500</td><td>Leu</td><td>pro</td><td>Leu</td><td>Val</td><td>Lys 505</td><td>asn</td><td>Arg</td><td>asn</td><td>ile</td><td>will 510</td><td>Ile</td><td>trp</td>

<td>Gly</td><td>thr</td><td>thr</td><td>Leu</td><td>tyr</td><td>pro</td><td>Lys</td><td>tyr</td><td>will</td><td>asn</td><td>Lys</td><td>val</td><td>asp</td><td>asn</td><td>pro</td><td>Ile</td>

515 520 525

Glu &lt;210&gt; 18 &lt;211&gt; 441 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 18

Met Lys Leu Lys Met val ile ile Phe Leu Leu Ile Leu Thr will val 15 10 15

Pro ile Ala Gin Gin Ala Glu Lys Thr Asp Ala Ala Phe will ASN Lys 20 25 30

Glu Ile ASN will will will val Ala Ala Pro Pro Pro Pro Ala will be 35 40 45

<td rowspan="2">pro</td><td colspan="7">Lys Thr Lys Pro Glu Ile Lys</td><td rowspan="2">His</td><td rowspan="2">Ala</td><td rowspan="2">asp</td><td rowspan="2">glu 60</td><td colspan="2" rowspan="2">ile Asp</td><td rowspan="2">Lys</td><td rowspan="2">tyr</td>

<td colspan="2"> 50</td><td colspan="5"> 55</td>

<td>Ile</td><td>gin</td><td rowspan="2">Gly</td><td>Leu</td><td rowspan="2">asp</td><td>tyr</td><td>asn</td><td rowspan="2">Lys</td><td>asn</td><td>asn</td><td>val</td><td>Leu</td><td>val</td><td rowspan="2">tyr</td><td>His</td><td>Gly</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>asp</td><td>Ala</td><td>val</td><td>thr</td><td>asn</td><td>Val</td><td>pro</td><td>pro</td><td rowspan="2">Arg</td><td>Lys</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td rowspan="2">Lys</td><td>asp</td><td>Gly</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td> 95</td><td></td>

<td>glu</td><td rowspan="2">tyr</td><td>ile</td><td>val</td><td>Val</td><td>glu</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Lys</td><td rowspan="2">Lys</td><td>will</td><td>Ile</td><td>asn</td><td>gin</td><td>asn</td><td>asn</td>

<td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ala</td><td rowspan="2">asp</td><td>Ile</td><td>gin</td><td>Val</td><td>Val</td><td>asn</td><td>Ala</td><td>ile</td><td>will</td><td>will</td><td>Leu</td><td>thr</td><td rowspan="2">tyr</td><td>pro</td><td rowspan="2">Gly</td>

<td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td>

<td>Ala</td><td>Leu</td><td>val</td><td rowspan="2">Lys</td><td>Ala</td><td>asn</td><td>will</td><td>glu</td><td>Leu</td><td>val</td><td>glu</td><td>asn</td><td>n gl</td><td>pro</td><td rowspan="2">asp</td><td>Val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>

<td>Leu</td><td>pro</td><td>Val</td><td>Lys</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Ile</td><td rowspan="2">asp</td><td>Leu</td><td>pro</td><td>Gly</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Met</td><td>thr</td><td>asn</td><td>gin</td><td>asp</td><td>asn</td><td rowspan="2">Lys</td><td>Ile</td><td>Val</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>Ala</td><td>thr</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asn</td><td>val</td><td>asn</td><td>asn</td><td>Ala</td><td>val</td><td>asn</td><td>thr</td><td>Leu</td><td>Val</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>asn</td><td>glu</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>tyr</td><td>Ala</td><td>gin</td><td>Ala</td><td>tyr</td><td>will</td><td>asn</td><td>val</td><td>will</td><td>Ala</td><td rowspan="2">Lys</td><td>ile</td><td>asp</td><td>tyr</td><td>asp</td><td>asp</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>glu</td><td>Met</td><td>Ala</td><td rowspan="2">tyr</td><td>will</td><td>glu</td><td>will</td><td>gin</td><td>Leu</td><td>Ile</td><td>Ala</td><td>Lys</td><td>Phe</td><td rowspan="2">Gly</td><td>thr</td><td>Ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>val</td><td>asn</td><td>asn</td><td>will</td><td>Leu</td><td>asn</td><td>val</td><td>asn</td><td>Phe</td><td rowspan="2">Gly</td><td>Ala</td><td>Ile</td><td>will</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>glu</td><td>Gly</td><td rowspan="2">Lys</td><td>Met</td><td>gin</td><td>glu</td><td>glu</td><td>val</td><td>Ile</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>gin</td><td>Ile</td><td>tyr</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td>

<td>asn</td><td>val</td><td>asn</td><td>val</td><td>asn</td><td>glu</td><td>pro</td><td>thr</td><td>Arg</td><td>pro</td><td>will</td><td rowspan="2">Arg</td><td>Phe</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>Ala</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td><td>gin</td><td>Leu</td><td>gin</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>Val</td><td>asn</td><td>Ala</td><td>glu</td><td>asn</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>pro</td><td>Ala</td><td rowspan="2">tyr</td><td>Ile</td><td>will</td><td>will</td><td>val</td><td>Ala</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Arg</td><td>gin</td><td>Val</td><td rowspan="2">tyr</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>will</td><td>thr</td><td>asn</td><td>will</td><td>His</td><td>will</td><td>thr</td><td>Lys</td><td>val</td><td>Lys</td><td>Ala</td><td>Ala</td><td>Phe</td><td>asp</td>

305 310 315 320

<td>Ala</td><td>Ala</td><td>val</td><td colspan="2">Will Gly 325</td><td colspan="3">Will Val Lys</td><td>will</td><td>Gly 330</td><td colspan="4">Leu Glu Asp val</td><td>thr 335</td><td>asn</td>

<td>Ile</td><td>Ile</td><td rowspan="2">Lys</td><td>asn</td><td>will</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>val</td><td>ile</td><td rowspan="2">tyr</td><td colspan="2">Gly Gly</td><td>will</td><td>Ala</td>

<td></td><td></td><td> 340</td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Lys</td><td>asp</td><td>glu</td><td>val</td><td>gin</td><td>ile</td><td>ile</td><td>asp</td><td>Gly</td><td>asn</td><td>Leu</td><td rowspan="2">Gly</td><td>asp</td><td>Leu</td><td rowspan="2">Arg</td><td>asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td> 365</td><td></td><td></td>

<td>ile</td><td>Leu</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td rowspan="2">Gly</td><td>Ala</td><td>thr</td><td>Phe</td><td>asn</td><td rowspan="2">Arg</td><td>glu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td>val</td><td>pro</td>

<td></td><td> 370</td><td></td><td> 375</td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td>

<td>ile</td><td>Ala</td><td rowspan="2">tyr</td><td>thr</td><td>thr</td><td>asn</td><td>Phe</td><td>Leu</td><td rowspan="2">Lys</td><td rowspan="2">asp</td><td>asn</td><td>glu</td><td>Leu</td><td>Ala</td><td>val</td><td>ile</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Lys</td><td>asn</td><td>asn</td><td>will</td><td>glu</td><td rowspan="2">tyr</td><td>ile</td><td>glu</td><td>thr</td><td>thr</td><td>will</td><td rowspan="2">Lys</td><td>Ala</td><td rowspan="2">tyr</td><td>thr</td><td>ASP</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td> 415</td><td></td>

<td>Gly</td><td>Lys</td><td>ile</td><td>asn</td><td>Ile</td><td>asp</td><td>His</td><td>will</td><td>Gly</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td>val</td><td>Ala</td><td>gin</td><td>Phe</td><td>asn</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td> 430</td><td></td><td></td>

<td>ile</td><td>will</td><td>trp</td><td>asp</td><td>glu</td><td>val</td><td>asn</td><td>tyr</td><td rowspan="2">asp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 19 &lt;211&gt; 416 &lt;212&gt; PRT &lt;213&gt; Listeria 1isteria &lt;400&gt; 19

<td>Met 1</td><td>Lys</td><td>Lys</td><td>Ile</td><td>Met 5</td><td>Leu</td><td>val</td><td>Phe</td>

<td>pro</td><td>ile</td><td>Ala</td><td>gin 20</td><td>gin</td><td>thr</td><td>glu</td><td>Ala</td>

<td>and Gl</td><td>asn</td><td>will 35</td><td>ile</td><td>will</td><td>will</td><td>val</td><td>Ala 40</td>

<td>pro</td><td>Lys 50</td><td>thr</td><td>pro</td><td>ile</td><td>glu</td><td>Lys 55</td><td>Lys</td>

<td>ile 65</td><td>gin</td><td>Gl y</td><td>Leu</td><td>asp</td><td>tyr 70</td><td>asn</td><td>Lys</td>

<td>asp</td><td>Ala</td><td>val</td><td>thr</td><td>asn 85</td><td>val</td><td>pro</td><td>pro</td>

<td>glu</td><td>tyr</td><td>Ile</td><td>val 100</td><td>val</td><td>glu</td><td>Lys</td><td>Lys</td>

<td>Ala</td><td>asp</td><td>Ile</td><td>gin</td><td>val</td><td>val</td><td>asn</td><td>Ala</td>

Leu Leu Thr Ile ile val will Leu 10 15

Lys Ala Phe Asp Ala will ASN Lys 25 30

Pro Pro Pro Pro Ala Ala Will Will 45

His Ala Glu Asp Tyr Lys Asp ile 60

Asn asn val val Tyr Leu Gly His 75 80

Lys Arg Gly Gly Tyr Lys Asp ASN 90 95

Lys Lys Gin will ile NSA NSA NSA 105 110

Ile Leu Thr will be pro Tyr Gly

-83115 120 125

<td colspan="3">Ala Val Leu</td><td colspan="3" rowspan="2">Lys Ala ASN</td><td colspan="4" rowspan="2">Val will Leu Glu 135</td><td colspan="2" rowspan="2">Glu ASN 140</td><td rowspan="2">gin</td><td rowspan="2">pro</td><td rowspan="2">asp</td><td rowspan="2">Val</td>

<td></td><td colspan="2"> 130</td>

<td>Leu</td><td>pro</td><td>val</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Ile</td><td rowspan="2">asp</td><td>Leu</td><td>pro</td><td>Gly</td>

<td> 145</td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Met</td><td>thr</td><td>asn</td><td>gin</td><td>asp</td><td>asn</td><td rowspan="2">Lys</td><td>Ile</td><td>val</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>Ala</td><td>thr</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asn</td><td>val</td><td>asn</td><td>asn</td><td>Ala</td><td>Val</td><td>asn</td><td>thr</td><td>Leu</td><td>val</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>asn</td><td>glu</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>tyr</td><td>Ala</td><td>gin</td><td>Ala</td><td rowspan="2">tyr</td><td>will</td><td>asn</td><td>Val</td><td>will</td><td>Ala</td><td rowspan="2">Lys</td><td>ile</td><td>asp</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td rowspan="2">asp</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td>

<td>glu</td><td>Met</td><td>Ala</td><td rowspan="2">tyr</td><td>will</td><td>glu</td><td>will</td><td>gin</td><td>Leu</td><td>Ile</td><td>Ala</td><td>Lys</td><td>Phe</td><td rowspan="2">Gly</td><td>thr</td><td>Ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>val</td><td>asn</td><td>asn</td><td>will</td><td>Leu</td><td>asn</td><td>val</td><td>asn</td><td>Phe</td><td rowspan="2">Gly</td><td>for a</td><td>ile</td><td>will</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>glu</td><td>Gly</td><td rowspan="2">Lys</td><td>Met</td><td>gin</td><td>glu</td><td>glu</td><td>Val</td><td>ile</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>gin</td><td>ile</td><td>tyr</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td>

<td>asn</td><td>val</td><td>asn</td><td>val</td><td>asn</td><td>glu</td><td>pro</td><td>thr</td><td>Arg</td><td>pro</td><td>will</td><td rowspan="2">Arg</td><td>phe</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>Ala</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td><td>gin</td><td>Leu</td><td>gin</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>val</td><td>asn</td><td>Ala</td><td>glu</td><td>asn</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>pro</td><td>Ala</td><td>tyr</td><td>ile</td><td>will</td><td>will</td><td>val</td><td>Ala</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Arg</td><td>gin</td><td>val</td><td rowspan="2">tyr</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>will</td><td>thr</td><td>asn</td><td>will</td><td>His</td><td>will</td><td>thr</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Ala</td><td>Ala</td><td>Phe</td><td>asp</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td> 320</td>

<td>Ala</td><td>Ala</td><td>val</td><td>will</td><td>Gly</td><td rowspan="2">Lys</td><td>will</td><td>val</td><td>will</td><td>Gly</td><td rowspan="2">ASP</td><td>val</td><td>glu</td><td>Leu</td><td>thr</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>ile</td><td>ile</td><td>Lys</td><td>asn</td><td>will</td><td>will</td><td>Phe</td><td>Lys</td><td>Ala</td><td>Val</td><td>ile</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Gly</td><td>will</td><td>Ala</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Lys</td><td>asp</td><td>glu</td><td>Val</td><td>gin</td><td>Ile</td><td>Ile</td><td>asp</td><td rowspan="2">Gly</td><td>asn</td><td>Leu</td><td rowspan="2">Gly</td><td>asp</td><td>Leu</td><td rowspan="2">Arg</td><td rowspan="2">asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td> 365</td><td></td>

<td>ile</td><td>Leu</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Gly</td><td>Ala</td><td>thr</td><td>Phe</td><td>asn</td><td rowspan="2">Arg</td><td>glu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td>val</td><td>pro</td>

<td></td><td> 370</td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td>

<td>ile</td><td>Ala</td><td>tyr</td><td>thr</td><td>thr</td><td>asn</td><td>Phe</td><td>Leu</td><td>Lys</td><td>asp</td><td>asn</td><td>glu</td><td>Leu</td><td>Ala</td><td>val</td><td>Ile</td>

-84385 390 ' 395 " 400

ASN ASN will Lys Glu Tyr Glu Ile Lys Thr Thr Ala Tyr will Thr Asp 405 410 415 &lt;210&gt; 20 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Listeria Seeliger &lt;400&gt; 20

Val will Arg Glu Thr Pro ile will Ala Glu Glu Thr Pro Pro Pro will 15 10 15

Ala Pro Thr &lt;210&gt; 21 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 21

ASN will Lys Glu Ala Met will ile will Ala Pro Pro Pro Pro Ala Will 15 10 15

Will Pro Lys &lt;210&gt; 22 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 22 gcggatccca tggagataca cctac

-85 &lt;210&gt; 23 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 23 gcggatccca tggagataca cctac 25 &lt;210&gt; 24 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Ludzki papillomavirus &lt;400&gt; 24

His Ala Arg Tyr Phe Thr Val Ile ASN 1 5 &lt;210&gt; 25 &lt;211&gt; 261 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 25

<td>Met 1</td><td>trp</td><td>val</td><td>pro</td><td>val 5</td><td>val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu 10</td><td>will</td><td>val</td><td>thr</td><td>trp</td><td>ile 15</td><td>Gly</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu 20</td><td>ile</td><td>Leu</td><td>will</td><td>Arg</td><td>ile 25</td><td>Val</td><td>Gly</td><td>Gly</td><td>trp</td><td>glu 30</td><td>CYS</td><td>glu</td>

<td>Lys</td><td>His</td><td>will</td><td>gin</td><td>pro</td><td>trp</td><td>gin</td><td>val</td><td>Leu</td><td>Val</td><td>Ala</td><td>will</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Ala</td>

CYS Gly Leu Gly val val val val His Pro Gin Leu Thr Ala Ala TRP 50 55 60

<td>His</td><td>CYS</td><td>ile</td><td>Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Arg</td><td>It's</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>His</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td>Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>It's</td><td>will</td><td>Phe</td>

His Asp Pro Pro Tyr Leu Leu Leu Lys Met will ASN Arg Leu Phe Arg 100 105 110

<td>pro</td><td>Gly</td><td>asp</td><td>asp</td><td>will</td><td>will</td><td>His</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu</td><td>will</td><td>glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>pro</td><td>Ala</td><td>glu</td><td>Leu</td><td>thr</td><td>asp</td><td>Ala</td><td>val</td><td>Lys</td><td>val</td><td>Met</td><td>asp</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td>

130

135

140

Leu Gly Glu Ala Pro Thr Thr Gly CYS will Ala Tyr Gly TRP will Ile 145 150 155 160

<td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td>Lys</td><td>Lys</td><td>Leu</td><td>gin</td><td>CYS</td><td>Val</td><td>asp</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>val</td><td>ile</td><td>will</td><td>asn</td><td>asp</td><td>val</td><td>CYS</td><td>Ala</td><td>gin</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td>Lys</td><td>val</td>

180

185

190

Met Phe Leu Lys Thr Gly CYS Ala Gly Arg Gly Lys Thr TRP will Thr 195 200 205

<td>CYS</td><td>will</td><td>Gly</td><td>asp</td><td>will</td><td>Gly</td><td>Gly</td><td>pro</td><td>Leu val</td><td>CYS</td><td>asn</td><td>Gly</td><td>val</td><td>Leu</td><td>gin</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ile</td><td>thr</td><td>will</td><td>trp</td><td>Gly</td><td>will</td><td>glu</td><td>pro CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td>Arg</td><td>pro</td>

225

230

235

240

Will Tyr Leu Lys Thr Lys Arg Tyr His val val TRP Ile Lys Thr Asp 245 250 255

ASN val Ile Ala Pro 260

-87 &lt;210&gt; 26 &lt;211&gt; 5873 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 26

<td>ggtgtcttag</td><td>gcacactggt</td><td>cttggagtgc</td><td>aaaggatcta</td><td>ggcacgtgag</td><td>gctttgtatg</td><td> 60</td>

<td>aagaatcggg</td><td>gatcgtaccc</td><td>accccctgtt</td><td>tctgtttcat</td><td>cctgggcatg</td><td>tctcctctgc</td><td> 120</td>

<td>ctttgtcccc</td><td>tagatgaagt</td><td>ctccatgagc</td><td>tacaagggcc</td><td>tggtgcatcc</td><td>agggtgatct</td><td> 180</td>

<td>agtaattgca</td><td>gaacagcaag</td><td>tgctagctct</td><td>ccctcccctt</td><td>ccacagctct</td><td>gggtgtggga</td><td> 240</td>

<td>gggggttgtc</td><td>cagcctccag</td><td>cagcatgggg</td><td>agggccttgg</td><td>tcagcctctg</td><td>ggtgccagca</td><td> 300</td>

<td>gggcaggggc</td><td>ggagtcctgg</td><td>ggaatgaagg</td><td>ttttataggg</td><td>ctcctggggg</td><td>aggctcccca</td><td> 360</td>

<td>gccccaagct</td><td>taccacctgc</td><td>acccggagag</td><td>ctgtgtcacc</td><td>atgtgggtcc</td><td>cggttgtctt</td><td> 420</td>

<td>cctcaccctg</td><td>tccgtgacgt</td><td>ggattggtga</td><td>gaggggccat</td><td>ggttgggggg</td><td>atgcaggaga</td><td> 480</td>

<td>gggagccagc</td><td>cctgactgtc</td><td>aagctgaggc</td><td>tctttccccc</td><td>ccaacccagc</td><td>accccagccc</td><td> 540</td>

<td>agacagggag</td><td>ctgggctctt</td><td>ttctgtctct</td><td>cccagcccca</td><td>cttcaagccc</td><td>atacccccag</td><td> 600</td>

<td>tcccctccat</td><td>attgcaacag</td><td>tcctcactcc</td><td>cacaccaggt</td><td>ccccgctccc</td><td>tcccacttac</td><td> 660</td>

<td>cccagaactt</td><td>tcttcccatt</td><td>tgcccagcca</td><td>gctccctgct</td><td>cccagctgct</td><td>ttactaaagg</td><td> 720</td>

<td>ggaagttcct</td><td>gggcatctcc</td><td>gtgtttctct</td><td>ttgtggggct</td><td>caaaacctcc</td><td>aaggacctct</td><td> 780</td>

<td>ctcaatgcca</td><td>ttggttcctt</td><td>ggaccgtatc</td><td>actggtccat</td><td>ctcctgagcc</td><td>cctcaatcct</td><td> 840</td>

<td>atcacagtct</td><td>actgactttt</td><td>cccattcagc</td><td>tgtgagtgtc</td><td>caaccctatc</td><td>ccagagacct</td><td> 900</td>

<td>tgatgcttgg</td><td>cctcccaatc</td><td>ttgccctagg</td><td>atacccagat</td><td>gccaaccaga</td><td>cacctccttc</td><td> 960</td>

<td>tttcctagcc</td><td>aggctatctg</td><td>gcctgagaca</td><td>acaaatgggt</td><td>ccctcagtct</td><td>ggcaatggga</td><td> 1020</td>

<td>ctctgagaac</td><td>tcctcattcc</td><td>ctgactctta</td><td>gccccagact</td><td>cttcattcag</td><td>tggcccacat</td><td> 1080</td>

<td>tttccttagg</td><td>aaaaacatga</td><td>gcatccccag</td><td>ccacaactgc</td><td>cagctctctg</td><td>agtccccaaa</td><td> 1140</td>

<td>tctgcatcct</td><td>tttcaaaacc</td><td>taaaaacaaa</td><td>aagaaaaaca</td><td>aataaaacaa</td><td>aaccaactca</td><td> 1200</td>

<td>gaccagaact</td><td>gttttctcaa</td><td>cctgggactt</td><td>cctaaacttt</td><td>ccaaaacctt</td><td>cctcttccag</td><td> 1260</td>

<td>caactgaacc</td><td>tcgccataag</td><td>gcacttatcc</td><td>ctggttccta</td><td>gcacccctta</td><td>tcccctcaga</td><td> 1320</td>

<td>atccacaact</td><td>tgtaccaagt</td><td>ttcccttctc</td><td>ccagtccaag</td><td>accccaaatc</td><td>accacaaagg</td><td> 1380</td>

<td>acccaatccc</td><td>cagactcaag</td><td>atatggtctg</td><td>ggcgctgtct</td><td>tgtgtctcct</td><td>accctgatcc</td><td> 1440</td>

<td>ctgggttcaa</td><td>ctctgctccc</td><td>agagcatgaa</td><td>gcctctccac</td><td>cagcaccagc</td><td>caccaacctg</td><td> 1500</td>

<td>caaacctagg</td><td>gaagattgac</td><td>agaattccca</td><td>gcctttccca</td><td>gctccccctg</td><td>cccatgtccc</td><td> 1560</td>

<td>aggactccca</td><td>gccttggttc</td><td>tctgcccccg</td><td>tgtcttttca</td><td>aacccacatc</td><td>ctaaatccat</td><td> 1620</td>

<td>ctcctatccg</td><td>agtcccccag</td><td>ttccccctgt</td><td>caaccctgat</td><td>tcccctgatc</td><td>tagcaccccc</td><td> 1680</td>

<td>tctgcaggcg</td><td>ctgcgcccct</td><td>catcctgtct</td><td>cggattgtgg</td><td>gaggctggga</td><td>gtgcgagaag</td><td> 1740</td>

<td>cattcccaac</td><td>cctggcaggt</td><td>gcttgtggcc</td><td>tctcgtggca</td><td>gggcagtctg</td><td>cggcggtgtt</td><td> 1800</td>

<td>ctggtgcacc</td><td>cccagtgggt</td><td>cctcacagct</td><td>gcccactgca</td><td>tcaggaagtg</td><td>agtaggggcc</td><td> 1860</td>

<td>tggggtctgg</td><td>ggagcaggtg</td><td>tctgtgtccc</td><td>agaggaataa</td><td>cagctgggca</td><td>ttttccccag</td><td> 1920</td>

<td>gataacctct</td><td>aaggccagcc</td><td>ttgggactgg</td><td>gggagagagg</td><td>gaaagttctg</td><td>gttcaggtca</td><td> 1980</td>

<td>catggggagg</td><td>cagggttggg</td><td>gctggaccac</td><td>cctccccatg</td><td>gctgcctggg</td><td>tctccatctg</td><td> 2040</td>

<td>tgtccctcta</td><td>tgtctctttg</td><td>tgtcgctttc</td><td>attatgtctc</td><td>ttggtaactg</td><td>gcttcggttg</td><td> 2100</td>

<td>tgtctctccg</td><td>tgtgactatt</td><td>ttgttctctc</td><td>tctccctctc</td><td>ttctctgtct</td><td>tcagtctcca</td><td> 2160</td>

<td>tatctccccc</td><td>tctctctgtc</td><td>cttctctggt</td><td>ccctctctag</td><td>ccagtgtgtc</td><td>tcaccctgta</td><td> 2220</td>

<td>tctctctgcc</td><td>aggctctgtc</td><td>tctcggtctc</td><td>tgtctcacct</td><td>gtgccttctc</td><td>cctactgaac</td><td> 2280</td>

<td>acacgcacgg</td><td>gatgggcctg</td><td>ggggaccctg</td><td>agaaaaggaa</td><td>gggctttggc</td><td>tgggcgcggt</td><td> 2340</td>

<td>ggctcacacc</td><td>tgtaatccca</td><td>gcactttggg</td><td>aggccaaggc</td><td>aggtagatca</td><td>cctgaggtca</td><td> 2400</td>

<td>ggagttcgag</td><td>accagcctgg</td><td>ccaactggtg</td><td>aaaccccatc</td><td>tctactaaaa</td><td>atacaaaaaa</td><td> 2460</td>

<td>ttagccaggc</td><td>gtggtggcgc</td><td>atgcctgtag</td><td>tcccagctac</td><td>tcaggagctg</td><td>agggaggaga</td><td> 2520</td>

<td>attgcattga</td><td>acctggaggt</td><td>tgaggttgca</td><td>gtgagccgag</td><td>accgtgccac</td><td>tgcactccag</td><td> 2580</td>

<td>cctgggtgac</td><td>agagtgagac</td><td>tccgcctcaa</td><td>aaaaaaaaaa</td><td>aaaaaaaaaa</td><td>aaaaaaaaga</td><td> 2640</td>

<td>aaagaaaaga</td><td>aaagaaaagg</td><td>aagtgtttta</td><td>tccctgatgt</td><td>gtgtgggtat</td><td>gagggtatga</td><td> 2700</td>

<td>gagggcccct</td><td>ctcactccat</td><td>tccttctcca</td><td>ggacatccct</td><td>ccactcttgg</td><td>gagacacaga</td><td> 2760</td>

<td>gaagggctgg</td><td>ttccagctgg</td><td>agctgggagg</td><td>ggcaattgag</td><td>ggaggaggaa</td><td>ggagaagggg</td><td> 2820</td>

<td>gaaggaaaac</td><td>agggtatggg</td><td>ggaaaggacc</td><td>ctggggagcg</td><td>aagtggagga</td><td>tacaaccttg</td><td> 2880</td>

<td>ggcctgcagg</td><td>caggctacct</td><td>acccacttgg</td><td>aaacccacgc</td><td>caaagccgca</td><td>tctacagctg</td><td> 2940</td>

<td>agccactctg</td><td>aggcctcccc</td><td>tccccggcgg</td><td>tccccactca</td><td>gctccaaagt</td><td>ctctctccct</td><td> 3000</td>

<td>tttctctccc</td><td>acactttatc</td><td>atcccccgga</td><td>ttcctctcta</td><td>cttggttctc</td><td>attcttcctt</td><td> 3060</td>

<td>tgacttcctg</td><td>cttccctttc</td><td>tcattcatct</td><td>gtttctcact</td><td>ttctgcctgg</td><td>ttttgttctt</td><td> 3120</td>

<td>ctctctctct</td><td>ttctctggcc</td><td>catgtctgtt</td><td>tctctatgtt</td><td>tctgtctttt</td><td>ctttctcatc</td><td> 3180</td>

<td>ctgtgtattt</td><td>tcggctcacc</td><td>ttgtttgtca</td><td>ctgttctccc</td><td>ctctgccctt</td><td>tcattctctc</td><td> 3240</td>

<td>tgccctttta</td><td>ccctcttcct</td><td>tttcccttgg</td><td>ttctctcagt</td><td>tctgtatctg</td><td>cccttcaccc</td><td> 3300</td>

<td>tctcacactg</td><td>ctgtttccca</td><td>actcgttgtc</td><td>tgtattttgg</td><td>cctgaactgt</td><td>gtcttcccaa</td><td> 3360</td>

<td>ccctgtgttt</td><td>tctcactgtt</td><td>tctttttctc</td><td>ttttggagcc</td><td>tcctccttgc</td><td>tcctctgtcc</td><td> 3420</td>

<td>cttctctctt</td><td>tccttatcat</td><td>cctcgctcct</td><td>cattcctgcg</td><td>tctgcttcct</td><td>ccccagcaaa</td><td> 3480</td>

<td>agcgtgatct</td><td>tgctgggtcg</td><td>gcacagcctg</td><td>tttcatcctg</td><td>aagacacagg</td><td>ccaggtattt</td><td> 3540</td>

<td>caggtcagcc</td><td>acagcttccc</td><td>acacccgctc</td><td>tacgatatga</td><td>gcctcctgaa</td><td>gaatcgattc</td><td> 3600</td>

<td>ctcaggccag</td><td>gtgatgactc</td><td>cagccacgac</td><td>ctcatgctgc</td><td>tccgcctgtc</td><td>agagcctgcc</td><td> 3660</td>

<td>gagctcacgg</td><td>atgctgtgaa</td><td>ggtcatggac</td><td>ctgcccaccc</td><td>aggagccagc</td><td>actggggacc</td><td> 3720</td>

<td>acctgctacg</td><td>cctcaggctg</td><td>gggcagcatt</td><td>gaaccagagg</td><td>agtgtacgcc</td><td>tgggccagat</td><td> 3780</td>

<td>ggtgcagccg</td><td>ggagcccaga</td><td>tgcctgggtc</td><td>tgagggagga</td><td>ggggacagga</td><td>ctcctgggtc</td><td> 3840</td>

<td>tgagggagga</td><td>gggccaagga</td><td>accaggtggg</td><td>gtccagccca</td><td>caacagtgtt</td><td>tttgcctggc</td><td> 3900</td>

<td>ccgtagtctt</td><td>gaccccaaag</td><td>aaacttcagt</td><td>gtgtggacct</td><td>ccatgttatt</td><td>tccaatgacg</td><td> 3960</td>

<td>tgtgtgcgca</td><td>agttcaccct</td><td>cagaaggtga</td><td>ccaagttcat</td><td>gctgtgtgct</td><td>ggacgctgga</td><td> 4020</td>

<td>cagggggcaa</td><td>aagcacctgc</td><td>tcggtgagtc</td><td>atccctactc</td><td>ccaagatctt</td><td>gagggaaagg</td><td> 4080</td>

<td>tgagtgggac</td><td>cttaattctg</td><td>ggctggggtc</td><td>tagaagccaa</td><td>caaggcgtct</td><td>gcctcccctg</td><td> 4140</td>

<td>ctccccagct</td><td>gtagccatgc</td><td>cacctccccg</td><td>tgtctcatct</td><td>cattccctcc</td><td>ttccctcttc</td><td> 4200</td>

<td>tttgactccc</td><td>tcaaggcaat</td><td>aggttattct</td><td>tacagcacaa</td><td>ctcatctgtt</td><td>cctgcgttca</td><td> 4260</td>

<td>gcacacggtt</td><td>actaggcacc</td><td>tgctatgcac</td><td>ccagcactgc</td><td>cctagagcct</td><td>gggacatagc</td><td> 4320</td>

<td>agtgaacaga</td><td>cagagagcag</td><td>cccctccctt</td><td>ctgtagcccc</td><td>caagccagtg</td><td>aggggcacag</td><td> 4380</td>

<td>gcaggaacag</td><td>ggaccacaac</td><td>acagaaaagc</td><td>tggagggtgt</td><td>caggaggtga</td><td>tcaggctctc</td><td> 4440</td>

<td>ggggagggag</td><td>aaggggtggg</td><td>gagtgtgact</td><td>gggaggagac</td><td>atcctgcaga</td><td>aggtgggagt</td><td> 4500</td>

<td>gagcaaacac</td><td>ctgcgcaggg</td><td>gaggggaggg</td><td>cctgcggcac</td><td>ctgggggagc</td><td>agagggaaca</td><td> 4560</td>

<td>gcatctggcc</td><td>aggcctggga</td><td>ggaggggcct</td><td>agagggcgtc</td><td>aggagcagag</td><td>aggaggttgc</td><td> 4620</td>

<td>ctggctggag</td><td>tgaaggatcg</td><td>gggcagggtg</td><td>cgagagggaa</td><td>caaaggaccc</td><td>ctcctgcagg</td><td> 4680</td>

<td>gcctcacctg</td><td>ggccacagga</td><td>ggacactgct</td><td>tttcctctga</td><td>ggagtcagga</td><td>actgtggatg</td><td> 4740</td>

<td>gtgctggaca</td><td>gaagcaggac</td><td>agggcctggc</td><td>tcaggtgtcc</td><td>agaggctgcg</td><td>ctggcctcct</td><td> 4800</td>

<td>atgggatcag</td><td>actgcaggga</td><td>gggagggcag</td><td>cagggatgtg</td><td>gagggagtga</td><td>tgatggggct</td><td> 4860</td>

<td>gacctggggg</td><td>tggctccagg</td><td>cattgtcccc</td><td>acctgggccc</td><td>ttacccagcc</td><td>tccctcacag</td><td> 4920</td>

<td>gctcctggcc</td><td>ctcagtctct</td><td>cccctccact</td><td>ccattctcca</td><td>cctacccaca</td><td>gtgggtcatt</td><td> 4980</td>

<td>ctgatcaccg</td><td>aactgaccat</td><td>gccagccctg</td><td>ccgatggtcc</td><td>tccatggctc</td><td>cctagtgccc</td><td> 5040</td>

<td>tggagaggag</td><td>gtgtctagtc</td><td>agagagtagt</td><td>cctggaaggt</td><td>ggcctctgtg</td><td>aggagccacg</td><td> 5100</td>

<td>gggacagcat</td><td>cctgcagatg</td><td>gtcctggccc</td><td>ttgtcccacc</td><td>gacctgtcta</td><td>caaggactgt</td><td> 5160</td>

<td>cctcgtggac</td><td>cctcccctct</td><td>gcacaggagc</td><td>tggaccctga</td><td>agtcccttcc</td><td>taccggccag</td><td> 5220</td>

<td>gactggagcc</td><td>cctacccctc</td><td>tgttggaatc</td><td>cctgcccacc</td><td>ttcttctgga</td><td>agtcggctct</td><td> 5280</td>

<td>ggagacattt</td><td>ctctcttctt</td><td>ccaaagctgg</td><td>gaactgctat</td><td>ctgttatctg</td><td>cctgtccagg</td><td> 5340</td>

<td>tctgaaagat</td><td>aggattgccc</td><td>aggcagaaac</td><td>tgggactgac</td><td>ctatctcact</td><td>ctctccctgc</td><td> 5400</td>

<td>ttttaccctt</td><td>agggtgattc</td><td>tgggggccca</td><td>cttgtctgta</td><td>atggtgtgct</td><td>tcaaggtatc</td><td> 5460</td>

<td>acgtcatggg</td><td>gcagtgaacc</td><td>atgtgccctg</td><td>cccgaaaggc</td><td>cttccctgta</td><td>caccaaggtg</td><td> 5520</td>

<td>gtgcattacc</td><td>ggaagtggat</td><td>caaggacacc</td><td>atcgtggcca</td><td>acccctgagc</td><td>acccctatca</td><td> 5580</td>

<td>agtccctatt</td><td>gtagtaaact</td><td>tggaaccttg</td><td>gaaatgacca</td><td>ggccaagact</td><td>caagcctccc</td><td> 5640</td>

-90cagttctact gacctttgtc cttaggtgtg aggtccaggg ttgctaggaa aagaaatcag 5700 cagacacagg tgtagaccag agtgtttctt aaatggtgta attttgtcct ctctgtgtcc 5760 tggggaatac tggccatgcc tggagacata tcactcaatt tctctgagga cacagttagg 5820 atggggtgtc tgtgttattt gtgggataca gagatgaaag aggggtggga tcc 5873 &lt;210&gt; 27 &lt;211&gt; 238 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 27

<td>Met</td><td rowspan="2">trp</td><td>val</td><td>pro</td><td>Val</td><td>val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Val</td><td>thr</td><td rowspan="2">trp</td><td>Ile</td><td rowspan="2">Gly</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>Ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>Ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td rowspan="2">Lys</td><td>His</td><td>will</td><td>gin</td><td>pro</td><td rowspan="2">trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td>Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>val</td><td>CYS</td><td>Gly</td><td>Gly</td><td>val</td><td>Leu</td><td>val</td><td>His</td><td>pro</td><td>gin</td><td>trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

55 60

<td>It's</td><td>CYS</td><td>ile</td><td>Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Arg</td><td>It's</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>It's</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td>Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>It's</td><td>will</td><td>Phe</td>

90 95

His Asp Pro Pro Tyr Leu Leu Leu Lys Met will ASN Arg Leu Phe Arg 100 105 110

<td>pro</td><td>Gly</td><td>asp</td><td>asp</td><td>will</td><td>will</td><td>It's</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu</td><td>will</td><td>glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>pro</td><td>Ala</td><td>glu</td><td>Leu</td><td>thr</td><td>asp</td><td>Ala</td><td>val</td><td>Lys</td><td>val</td><td>Met</td><td>asp</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td>

130 135 140

Leu Gly Glu Ala Pro Thr Thr Gly TRP will CYS Tyr Ala Gly will ile 145 150 155 160

<td colspan="4">Pro Glu Glu Glu</td><td>Phe 165</td><td colspan="2">Leu thr</td><td colspan="2">pro Lys</td><td>Lys 170</td><td>Leu</td><td>gin</td><td>CYS</td><td>Val</td><td>asp 175</td><td>Leu</td>

<td>It's</td><td>val</td><td>ile</td><td>will</td><td>asn</td><td rowspan="2">asp</td><td>val</td><td rowspan="2">CYS</td><td>Ala</td><td>gin</td><td>val</td><td>His</td><td>pro</td><td>gin</td><td rowspan="2">Lys</td><td>Val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td>

<td>thr</td><td>Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td rowspan="2">Arg</td><td>trp</td><td>thr</td><td>Gly</td><td>Gly</td><td>Lys</td><td>will</td><td>thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

CYS will TRP val Ile Leu Thr Ile Glu Thr Met Leu Leu Ala Pro Pro 210 215 220

His Met Leu Gly val val will Leu Gly Arg Gly Pro TRP val 225 230 235 &lt;210&gt; 28 &lt;211&gt; 1906 &lt;212&gt; DNA &lt;213&gt; Homo sapiens

-92&lt;400&gt; 28

<td>agccccaagc</td><td>ttaccacctg</td><td>cacccggaga</td><td>gctgtgtcac</td><td>catgtgggtc</td><td>ccggttgtct</td><td> 60</td>

<td>tcctcaccct</td><td>gtccgtgacg</td><td>tggattggtg</td><td>ctgcacccct</td><td>catcctgtct</td><td>cggattgtgg</td><td> 120</td>

<td>gaggctggga</td><td>gtgcgagaag</td><td>cattcccaac</td><td>cctggcaggt</td><td>gcttgtggcc</td><td>tctcgtggca</td><td> 180</td>

<td>gggcagtctg</td><td>cggcggtgtt</td><td>ctggtgcacc</td><td>cccagtgggt</td><td>cctcacagct</td><td>gcccactgca</td><td> 240</td>

<td>tcaggaacaa</td><td>aagcgtgatc</td><td>ttgctgggtc</td><td>ggcacagcct</td><td>gtttcatcct</td><td>gaagacacag</td><td> 300</td>

<td>nrra a 1- η n + L3 --- 33 - - -</td><td>tranntranr</td><td>raranrt + kk ^ r V.</td><td>γώγώγγγπγ + k.</td><td>V. cousin kij</td><td>3 / the γΥγγ1- / ίώ</td><td>w</td>

<td>agaatcgatt</td><td>cctcaggcca</td><td>ggtgatgact</td><td>ccagccacga</td><td>cctcatgctg</td><td>ctccgcctgt</td><td> 420</td>

<td>cagagcctgc</td><td>cgagctcacg</td><td>gatgctgtga</td><td>aggtcatgga</td><td>cctgcccacc</td><td>caggagccag</td><td> 480</td>

<td>cactggggac</td><td>cacctgctac</td><td>gcctcaggct</td><td>ggggcagcat</td><td>tgaaccagag</td><td>gagttcttga</td><td> 540</td>

<td>ccccaaagaa</td><td>acttcagtgt</td><td>gtggacctcc</td><td>atgttatttc</td><td>caatgacgtg</td><td>tgtgcgcaag</td><td> 600</td>

<td>ttcaccctca</td><td>gaaggtgacc</td><td>aagttcatgc</td><td>tgtgtgctgg</td><td>acgctggaca</td><td>gggggcaaaa</td><td> 660</td>

<td>gcacctgctc</td><td>gtgggtcatt</td><td>ctgatcaccg</td><td>aactgaccat</td><td>gccagccctg</td><td>ccgatggtcc</td><td> 720</td>

<td>tccatggctc</td><td>cctagtgccc</td><td>tggagaggag</td><td>gtgtctagtc</td><td>agagagtagt</td><td>cctggaaggt</td><td> 780</td>

<td>ggcctctgtg</td><td>aggagccacg</td><td>gggacagcat</td><td>cctgcagatg</td><td>gtcctggccc</td><td>ttgtcccacc</td><td> 840</td>

<td>gacctgtcta</td><td>caaggactgt</td><td>cctcgtggac</td><td>cctcccctct</td><td>gcacaggagc</td><td>tggaccctga</td><td> 900</td>

<td>agtcccttcc</td><td>ccaccggcca</td><td>ggactggagc</td><td>ccctacccct</td><td>ctgttggaat</td><td>ccctgcccac</td><td> 960</td>

<td>cttcttctgg</td><td>aagtcggctc</td><td>tggagacatt</td><td>tctctcttct</td><td>tccaaagctg</td><td>ggaactgcta</td><td> 1020</td>

<td>tctgttatct</td><td>gcctgtccag</td><td>gtctgaaaga</td><td>taggattgcc</td><td>caggcagaaa</td><td>ctgggactga</td><td> 1080</td>

<td>cctatctcac</td><td>tctctccctg</td><td>cttttaccct</td><td>tagggtgatt</td><td>ctgggggccc</td><td>acttgtctgt</td><td> 1140</td>

<td>aatggtgtgc</td><td>ttcaaggtat</td><td>cacgtcatgg</td><td>ggcagtgaac</td><td>catgtgccct</td><td>gcccgaaagg</td><td> 1200</td>

<td>ccttccctgt</td><td>acaccaaggt</td><td>ggtgcattac</td><td>cggaagtgga</td><td>tcaaggacac</td><td>catcgtggcc</td><td> 1260</td>

<td>------- aarrrrtnan 3-3</td><td>cacccctatc</td><td>aaccccctat</td><td>tntantaaar -3--3 -----</td><td>ttnnaarrtt --33 ------</td><td>nnaaatnarr 33- - -</td><td> 1320</td>

<td>aggccaagac</td><td>tcaagcctcc</td><td>ccagttctac</td><td>tgacctttgt</td><td>ccttaggtgt</td><td>gaggtccagg</td><td> 1380</td>

<td>gttgctagga</td><td>aaagaaatca</td><td>gcagacacag</td><td>gtgtagacca</td><td>gagtgtttct</td><td>taaatggtgt</td><td> 1440</td>

<td>aattttgtcc</td><td>tctctgtgtc</td><td>ctggggaata</td><td>ctggccatgc</td><td>ctggagacat</td><td>atcactcaat</td><td> 1500</td>

<td>ttctctgagg</td><td>acacagatag</td><td>gatggggtgt</td><td>ctgtgttatt</td><td>tgtggggtac</td><td>agagatgaaa</td><td> 1560</td>

<td>gaggggtggg</td><td>atccacactg</td><td>agagagtgga</td><td>gagtgacatg</td><td>tgctggacac</td><td>tgtccatgaa</td><td> 1620</td>

gcactgagca gaagctggag gcacaacgca ccagacactc acagcaagga tggagctgaa 1680 aacataaccc actctgtcct ggaggcactg ggaagcctag agaaggctgt gagccaagga 1740 gggagggtct tcctttggca tgggatgggg atgaagtaag gagagggact ggaccccctg 1800 gaagctgatt cactatgggg ggaggtgtat tgaagtcctc cagacaaccc tcagatttga 1860 tgatttccta gtagaactca cagaaataaa gagctgttat actgtg. 1906 &lt;210&gt; 29

-93 &lt;211&gt; 69 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 29

<td>Met</td><td rowspan="2">trp</td><td>val</td><td>pro</td><td>Val</td><td>Val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>val</td><td>thr</td><td rowspan="2">trp</td><td>ile</td><td rowspan="2">Gly</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>Ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td>Lys</td><td>It's</td><td>will</td><td>gin</td><td>pro</td><td rowspan="2">trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>val</td><td>CYS</td><td>Gly</td><td>Gly</td><td>val</td><td>Leu</td><td>val</td><td>His</td><td>pro</td><td>gin</td><td>trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

55 60

His CYS Arg Ile Lys 65 &lt;210&gt; 30 &lt;211&gt; 220 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

<td colspan="8"> &lt;400&gt; 30</td><td rowspan="2">thr</td><td rowspan="2">Leu 10</td><td rowspan="2">will</td><td rowspan="2">val</td><td colspan="2" rowspan="2">thr TRP</td><td rowspan="2">Ile 15</td><td rowspan="2">Gly</td>

<td colspan="2">Met TRP 1</td><td>val</td><td colspan="2">pro val 5</td><td>val</td><td>Phe</td><td>Leu</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>Ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>Ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td>Lys</td><td>It's</td><td>will</td><td>gin</td><td>pro</td><td rowspan="2">trp</td><td>n gl</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>val</td><td>CYS</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>val</td><td>Leu</td><td>val</td><td>His</td><td>pro</td><td>gin</td><td rowspan="2">trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>It's</td><td>CYS</td><td>Ile</td><td>Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>His</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Phe</td><td>His</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td rowspan="2">Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>His</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>pro</td><td>His</td><td>pro</td><td>Leu 100</td><td colspan="4">Tyr Met Asp will</td><td colspan="3">Leu Leu Lys 105</td><td colspan="2">asn Arg</td><td>Phe 110</td><td>Leu</td><td>Arg</td>

<td>pro</td><td>Gly</td><td>asp</td><td rowspan="2">ASP</td><td>will</td><td>will</td><td>Ile</td><td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Lys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>gin</td><td rowspan="2">CYS</td><td>val</td><td rowspan="2">asp</td><td>Leu</td><td>His</td><td>val</td><td>ile</td><td>will</td><td>asn</td><td rowspan="2">ASP</td><td>Val</td><td rowspan="2">CYS</td><td>for a</td>

<td></td><td> 130</td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td>

<td>gin</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td>Lys</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td rowspan="2">CYS</td><td>Ala</td><td rowspan="2">Gly</td><td>Arg</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td> 160</td>

<td>trp</td><td>thr</td><td>Gly</td><td>Gly</td><td>Lys</td><td>will</td><td>thr</td><td rowspan="2">CYS</td><td>will</td><td>Gly</td><td>asp</td><td>will</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>pro</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td rowspan="2">CYS</td><td>asn</td><td>Gly</td><td>val</td><td>Leu</td><td>gin</td><td rowspan="2">Gly</td><td>ile</td><td>thr</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>will</td><td>glu</td><td>pro</td>

<td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td><td></td>

<td>CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td rowspan="2">Arg</td><td>pro</td><td>will</td><td>Leu</td><td rowspan="2">tyr</td><td>thr</td><td rowspan="2">Lys</td><td>Val</td><td>val</td><td>His</td><td rowspan="2">tyr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td> 205</td><td></td><td></td>

<td>Arg</td><td>Lys</td><td>trp</td><td>Ile</td><td>Lys</td><td rowspan="2">asp</td><td>thr</td><td>ile</td><td>val</td><td>Ala</td><td>asn</td><td>pro</td><td></td><td></td><td></td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 31 &lt;211&gt; 218 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 31

<td>Met</td><td rowspan="2">trp</td><td>val</td><td>pro</td><td>val</td><td>Val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>val</td><td>thr</td><td rowspan="2">trp</td><td>ile</td><td rowspan="2">Gly</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td>Lys</td><td>His</td><td>will</td><td>gin</td><td>pro</td><td>trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>val</td><td>CYS</td><td>Gly</td><td>Gly</td><td>Val</td><td>Leu</td><td>Val</td><td>It's</td><td>pro</td><td>n gl</td><td>trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

55 60

<td>His 65</td><td>CYS</td><td>Ile</td><td>Arg</td><td>Lys</td><td>pro 70</td><td colspan="2">Gly Asp</td><td>asp</td><td>will</td><td>will 75</td><td colspan="2">His Asp</td><td>Leu</td><td>Met</td><td>Leu 80</td>

<td>Leu</td><td rowspan="2">Arg</td><td>Leu</td><td>will</td><td>glu</td><td>pro</td><td>Ala</td><td>glu</td><td>Leu</td><td>thr</td><td rowspan="2">asp</td><td>Ala</td><td>val</td><td rowspan="2">Lys</td><td>val</td><td>Met</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td> 95</td><td></td>

<td rowspan="2">asp</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td><td>glu</td><td>pro</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>thr</td><td>thr</td><td rowspan="2">CYS</td><td>tyr</td><td>Ala</td><td>will</td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td><td></td>

<td colspan="13">TRP will Gly Gly Glu Pro ile Phe Leu Glu Glu Thr Pro</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td rowspan="2">Leu</td>

<td></td><td colspan="5"> 115</td><td colspan="2"> 120</td><td colspan="5"> 125</td>

<td>gin</td><td>CYS</td><td>Val</td><td rowspan="2">asp</td><td>Leu</td><td>His</td><td>val</td><td>Ile</td><td>will</td><td>asn</td><td rowspan="2">asp</td><td>val</td><td rowspan="2">CYS</td><td>Ala</td><td>gin</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>

<td>It's</td><td>pro</td><td>gin</td><td>Lys</td><td>val</td><td>thr</td><td>Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td>Arg</td><td>trp</td><td>thr</td>

145 150 155 160

Gly Lys Thr Gly will CYS will be Gly Asp Gly Gly Val Leu Pro CYS 165 170 175

<td>asn</td><td>Gly</td><td>Val</td><td>Leu</td><td>gin</td><td rowspan="2">Gly</td><td>Ile</td><td>thr</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>will</td><td>glu</td><td>pro</td><td rowspan="2">CYS</td><td>Ala</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>Leu</td><td>pro</td><td>glu</td><td rowspan="2">Arg</td><td>pro</td><td>will</td><td>Leu</td><td>tyr</td><td>thr</td><td rowspan="2">Lys</td><td>val</td><td>val</td><td>His</td><td rowspan="2">tyr</td><td rowspan="2">Arg</td><td rowspan="2">Lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td>

<td rowspan="2">trp</td><td>Ile</td><td rowspan="2">Lys</td><td rowspan="2">asp</td><td>thr</td><td>ile</td><td>val</td><td>Ala</td><td>asn</td><td>pro</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 210</td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 32 &lt;211&gt; 261 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 32

<td>Met 1</td><td colspan="2">trp val</td><td>pro</td><td>val 5</td><td>val</td><td colspan="3">Phe Leu Thr</td><td colspan="2">Leu will 10</td><td>Val</td><td colspan="2">thr TRP</td><td>ile 15</td><td>Gly</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td>Lys</td><td>It's</td><td>will</td><td>gin</td><td>pro</td><td rowspan="2">trp</td><td>gin</td><td>Val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>val</td><td>CYS</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Val</td><td>Leu</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td rowspan="2">trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>His</td><td rowspan="2">CYS</td><td>Ile</td><td>Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>Ile</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>It's</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Phe</td><td>His</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td rowspan="2">Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>Val</td><td>will</td><td>His</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>pro</td><td>His</td><td>pro</td><td>Leu</td><td>tyr</td><td>asp</td><td>Met</td><td>will</td><td>Leu</td><td>Leu</td><td rowspan="2">Lys</td><td>asn</td><td rowspan="2">Arg</td><td>Phe</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td>

<td>pro</td><td>Gly</td><td>asp</td><td>asp</td><td>will</td><td>will</td><td>It's</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu</td><td>will</td><td>glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>pro</td><td>Ala 130</td><td>glu</td><td colspan="2">Leu thr</td><td>asp</td><td colspan="2">Ala val 135</td><td>Lys</td><td>Val '</td><td>Met</td><td>asp 140</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td>

<td>glu</td><td>pro</td><td>Ala</td><td>Leu</td><td>Gly</td><td>thr</td><td>thr</td><td>CYS</td><td>tyr</td><td>Ala</td><td>will</td><td rowspan="2">Gly</td><td>trp</td><td rowspan="2">Gly</td><td>will</td><td>ile</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td> 160</td>

<td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td>Lys</td><td>Lys</td><td>Leu</td><td>gin</td><td>CYS</td><td>Val</td><td>asp</td><td>Leu</td>

165 170 175

His Val ile will ASN Asp Val Ala CYS val His Gin Gin Lys Pro Val 180 185 190

<td>thr</td><td>Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>thr</td><td>Gly</td><td>Gly</td><td>Lys</td><td>will thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td> 205</td><td></td><td></td>

<td>CYS</td><td>will</td><td>Gly</td><td>asp</td><td>will</td><td>Gly</td><td>Gly</td><td>pro</td><td>Leu</td><td>val</td><td>CYS</td><td>asn</td><td>Gly</td><td>val</td><td>Leu Gin</td>

210 215' 220

<td>Gly</td><td>Ile</td><td>thr</td><td>will</td><td>trp</td><td>Gly</td><td>will</td><td>glu</td><td>pro</td><td>CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td>Arg</td><td>pro</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td>Leu</td><td>tyr</td><td>thr</td><td>Lys</td><td>val</td><td>val</td><td>His</td><td>tyr</td><td>Arg</td><td>Lys</td><td>trp</td><td>Ile</td><td>Lys</td><td>asp</td><td>thr</td>

245 250 255

ASN val Ile Ala Pro 260

-97 &lt;210&gt; 33 &lt;211&gt; 1464 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 33 agccccaagc ttaccacctg cacccggaga gctgtgtcac catgtgggtc ccggttgtct 60 tcctcaccct gtccgtgacg tggattggtg ctgcacccct catcctgtct cggattgtgg 120 gaggctggga gtgcgagaag cattcccaac cctggcaggt gcttgtggcc tctcgtggca 180 gggcagtctg cggcggtgtt ctggtgcacc cccagtgggt cctcacagct gcccactgca 240 tcaggaacaa aagcgtgatc ttgctgggtc ggcacagcct gtttcatcct gaagacacag 300 gccaggtatt tcaggtcagc cacagcttcc cacacccgct ctacgatatg agcctcctga 360 agaatcgatt cctcaggcca ggtgatgact ccagccacga cctcatgctg ctccgcctgt 420 cagagcctgc cgagctcacg gatgctgtga aggtcatgga cctgcccacc caggagccag 480 cactggggac cacctgctac gcctcaggct ggggcagcat tgaaccagag gagttcttga 540 ccccaaagaa acttcagtgt gtggacctcc atgttatttc caatgacgtg tgtgcgcaag 600 ttcaccctca gaaggtgacc aagttcatgc tgtgtgctgg acgctggaca gggggcaaaa 660 gcacctgctc gggtgattct gggggcccac ttgtctgtaa tggtgtgctt caaggtatca 720 cgtcatgggg cagtgaacca tgtgccctgc ccgaaaggcc ttccctgtac accaaggtgg 780 tgcattaccg gaagtggatc aaggacacca tcgtggccaa cccctgagca cccctatcaa 840 ccccctattg tagtaaactt ggaaccttgg aaatgaccag gccaagactc aagcctcccc 900 agttctactg acctttgtcc ttaggtgtga ggtccagggt tgctaggaaa agaaatcagc 960 agacacaggt gtagaccaga gtgtttctta aatggtgtaa ttttgtcctc tctgtgtcct 1020 ggggaatact ggccatgcct ggagacatat cactcaattt ctctgaggac acagatagga 1080 tggggtgtct gtgttatttg tggggtacag agatgaaaga ggggtgggat ccacactgag 1140 agagtggaga gtgacatgtg ctggacactg tccatgaagc actgagcaga agctggaggc 1200 acaacgcacc agacactcac agcaaggatg gagctgaaaa cataacccac tctgtcctgg 1260 aggcactggg aagcctagag aaggctgtga gccaaggagg gagggtcttc ctttggcatg 1320 ggatggggat gaagtaagga gagggactgg accccctgga agctgattca ctatgggggg 1380 aggtgtattg aagtcctcca gacaaccctc agatttgatg atttcctagt agaactcaca 1440 gaaataaaga gctgttatac tgtg 1464 &lt;210&gt; 34 &lt;211&gt; 261 &lt;212&gt; PRT

-98 &lt;213&gt; Homo sapiens

<td colspan="5"> &lt;400&gt; 34</td><td rowspan="2">val</td><td rowspan="2">Phe</td><td colspan="2" rowspan="2">Leu thr</td><td rowspan="2">Leu 10</td><td rowspan="2">will</td><td rowspan="2">val</td><td rowspan="2">thr</td><td rowspan="2">trp</td><td rowspan="2">ile 15</td><td rowspan="2">Gly</td>

<td>Met 1</td><td>trp</td><td>Val</td><td colspan="2">Val Pro 5</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>Ile</td><td>val</td><td rowspan="2">Gly</td><td>Gly</td><td>trp</td><td>glu</td><td>CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Lys</td><td>His</td><td>will</td><td>gin</td><td>pro</td><td>trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>Val</td><td>CYS</td><td colspan="2">Gly Gly</td><td>val</td><td>Leu</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td rowspan="2">trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>His</td><td>CYS</td><td>Ile</td><td rowspan="2">Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>His</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Phe</td><td>It's</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td rowspan="2">Gly</td><td>gin</td><td>Val</td><td>Phe</td><td>gin</td><td>Val</td><td>will</td><td>It's</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>pro</td><td>His</td><td>pro</td><td>Leu</td><td>tyr</td><td rowspan="2">asp</td><td>Met</td><td>will</td><td>Leu</td><td>Leu</td><td rowspan="2">Lys</td><td>asn</td><td rowspan="2">Arg</td><td>Phe</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td>

<td>pro</td><td rowspan="2">Gly</td><td>asp</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>His</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu</td><td>will</td><td>glu</td>

<td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>pro</td><td>Ala</td><td>and Gl</td><td>Leu</td><td>thr</td><td rowspan="2">asp</td><td>Ala</td><td>val</td><td rowspan="2">Lys</td><td>val</td><td>Met</td><td>asp</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td colspan="7">Pro Ala Gly Glu Thr Leu Thr</td><td colspan="3" rowspan="2">CYS Tyr Ala</td><td colspan="4" rowspan="2">TRP will Gly Gly 155</td><td rowspan="2">will</td><td rowspan="2">Ile 160</td>

<td colspan="3"> 145</td><td colspan="4"> 150</td>

<td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Lys</td><td>Lys</td><td>Leu</td><td>gin</td><td rowspan="2">CYS</td><td>Val</td><td>asp</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>Val</td><td>ile</td><td>will</td><td>asn</td><td rowspan="2">asp</td><td>val</td><td rowspan="2">CYS</td><td>Ala</td><td>gin</td><td>val</td><td>His</td><td>pro</td><td>gin</td><td rowspan="2">Lys</td><td>val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td>

<td>thr</td><td>Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>thr</td><td>Gly</td><td>Gly</td><td>Lys</td><td>will</td><td>thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td rowspan="2">CYS</td><td>will</td><td>Gly</td><td>asp</td><td>will</td><td>Gly</td><td>Gly</td><td>pro</td><td>Leu</td><td>Val</td><td rowspan="2">CYS</td><td>asn</td><td>Gly</td><td>val</td><td>Leu</td><td>gin</td>

<td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ile</td><td>thr</td><td>will</td><td rowspan="2">trp</td><td>Gly</td><td>will</td><td>glu</td><td>pro</td><td rowspan="2">CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td rowspan="2">Arg</td><td>pro</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td>Leu</td><td>tyr</td><td>thr</td><td>Lys</td><td>val</td><td>val</td><td>His</td><td>tyr</td><td>Arg</td><td>Lys</td><td>trp</td><td>ile</td><td>Lys</td><td>asp</td><td>thr</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

ile val ASN Pro 260 Ala &lt;210&gt; 35 &lt;211&gt; 1495 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 35

-100gggggagccc tgtcttcctc tgtgggaggc tggcagggca ctgcatcagg cacaggccag cctgaagaat cctgtcagag gccagcactg cttgacccca gcaagttcac caaaagcacc tatcacgtca ggtggtgcat atcaactccc tccccagttc tcagcagaca gtcctgggga taggatgggg ctgagagagt gaggcacaac cctggaggca gcatgggatg gggggaggtg tcacagaaat &lt;210&gt; 36 &lt;211&gt; 218 &lt;212&gt; PRT &lt;213&gt; Homo caagcttacc accctgtccg tgggagtgcg gtctgcggcg aacaaaagcg gtatttcagg cgattcctca cctgccgagc gggaccacct aagaaacttc cctcagaagg tgctcgggtg tggggcagtg taccggaagt tattgtagta tactgacctt caggtgtaga atactggcca tgtctgtgtt ggagagtgac gcaccagaca ctgggaagcc gggatgaagt tattgaagtc aaagagctgt acctgcaccc tgacgtggat agaagcattc gtgttctggt tgatcttgct tcagccacag ggccaggtga tcacggatgc gctacgcctc agtgtgtgga tgaccaagtt attctggggg aaccatgtgc ggatcaagga aacttggaac tgtccttagg ccagagtgtt tgcctggaga atttgtgggg atgtgctgga ctcacagcaa tagagaaggc agggagaggg ctccagacaa tatactgcga ggagagctgt tggtgctgca ccaaccctgg gcacccccag gggtcggcac cttcccacac tgactccagc tgtgaaggtc aggctggggc cctccatgtt catgctgtgt cccacttgtc cctgcccgaa caccatcgtg cttggaaatg tgtgaggtcc tcttaaatgg catatcactc tacagagatg cactgtccat ggatggagct tgtgagccaa actggacccc ccctcagatt aaaaaaaaaa gtcaccatgt cccctcatcc caggtgcttg tgggtcctca agcctgtttc ccgctctacg cacgacctca atggacctgc agcattgaac atttccaatg gctggacgct tgtaatggtg aggccttccc gccaacccct accaggccaa agggttgcta tgtaattttg aatttctctg aaagaggggt gaagcactga gaaaacataa ggagggaggg ctggaagctg tgatgatttc aaaaaaaaaa gggtcccggt tgtctcggat tggcctctcg cagctgccca atcctgaaga atatgagcct tgctgctccg ccacccagga cagaggagtt acgtgtgtgc ggacaggggg tgcttcaagg tgtacaccaa gagcacccct gactcaggcc ggaaaagaaa tcctctctgt aggacacaga gggatccaca gcagaagctg cccactctgt tcttcctttg attcactatg ctagtagaac aaaaa

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1495 sapiens &lt;400&gt; 36

-101-

<td colspan="2" rowspan="2">Met TRP 1</td><td rowspan="2">VA1</td><td rowspan="2">pro</td><td rowspan="2">val 5</td><td rowspan="2">val</td><td colspan="2" rowspan="2">Phe Leu</td><td colspan="7">Thr Leu Thr TRP will val ile</td><td rowspan="2">Gly</td>

<td colspan="3"> 10</td><td colspan="4"> 15</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>Ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td>

<td>Lys</td><td>It's</td><td>will</td><td>n gl</td><td>pro</td><td rowspan="2">trp</td><td>gin</td><td>Val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>val</td><td>CYS</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>val</td><td>Leu</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td rowspan="2">trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>It's</td><td>CYS</td><td>ile</td><td rowspan="2">Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>It's</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Phe</td><td>His</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td rowspan="2">Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>It's</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>ΡΓΟ</td><td>His</td><td>pro</td><td>Leu</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Met</td><td>will</td><td>Leu</td><td>Leu</td><td rowspan="2">Lys</td><td>asn</td><td rowspan="2">Arg</td><td>Phe</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td>

<td>pro</td><td>Gly</td><td>asp</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Ile</td><td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Lys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>gin</td><td>CYS</td><td>val</td><td>asp</td><td>Leu</td><td>It's</td><td>val</td><td>Ile</td><td>will</td><td>asn</td><td rowspan="2">asp</td><td>val</td><td rowspan="2">CYS</td><td>Ala</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td>

<td>gin</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td>Lys</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>Phe</td><td>Met</td><td>Leu</td><td rowspan="2">CYS</td><td>Ala</td><td rowspan="2">Gly</td><td>Arg</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td rowspan="2">Gly</td><td> 155</td><td></td><td></td><td> 160</td>

<td>trp</td><td>thr</td><td colspan="2">Gly Gly</td><td>Lys</td><td>will</td><td>thr</td><td rowspan="2">CYS</td><td>will</td><td>asp</td><td>will</td><td>Gly</td><td>Gly</td><td>pro</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td rowspan="2">CYS</td><td>asn</td><td>Gly</td><td>val</td><td>Leu</td><td>gin</td><td rowspan="2">Gly</td><td>Ile</td><td>thr</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>will</td><td>glu</td><td>pro</td>

<td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td><td></td>

<td>CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td rowspan="2">Arg</td><td>pro</td><td>will</td><td>Leu</td><td rowspan="2">tyr</td><td>thr</td><td>Lys</td><td>val</td><td>val</td><td>His</td><td rowspan="2">tyr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>Arg</td><td>Lys</td><td>trp</td><td>Ile</td><td>Lys</td><td>asp</td><td>thr</td><td>Ile</td><td>val</td><td>Ala</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 37 &lt;211&gt; 227 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

-102&lt;400&gt; 37

<td>Met 1</td><td>trp</td><td>val</td><td>pro</td><td>val 5</td><td>val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu 10</td><td>will</td><td>val</td><td>thr</td><td>trp</td><td>ile 15</td><td>Gly</td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>Ile</td><td>Leu</td><td>will</td><td>Arg</td><td>ile</td><td>Val</td><td>Gly</td><td>Gly</td><td>trp</td><td>glu</td><td>CYS</td><td>glu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Lys</td><td>It's</td><td>will</td><td>gin</td><td>pro</td><td>trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>CYS</td><td>Gly</td><td>Gly</td><td>val</td><td>Leu</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td>trp</td><td>val</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td>It's</td><td>CYS</td><td>ile</td><td>Arg</td><td>asn</td><td>Lys</td><td>will</td><td>val</td><td>ile</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Arg</td><td>His</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Phe</td><td>His</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td>Gly</td><td>gin</td><td>Val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>It's</td><td>will</td><td>Phe</td>

His Asp Pro Pro Tyr Leu Leu Leu Lys Met will ASN Arg Leu Phe Arg 100 105 110

<td>pro</td><td>Gly</td><td>asp</td><td>asp</td><td>will</td><td>will</td><td>His</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu will</td><td>glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>pro</td><td>Ala</td><td>and Gl</td><td>Leu</td><td>thr</td><td>asp</td><td>Ala</td><td>val</td><td>Lys</td><td>Val</td><td>Met</td><td>asp</td><td>Leu</td><td>pro thr</td><td>gin</td>

130

135

140

<td>glu</td><td>pro</td><td>Ala</td><td>Leu</td><td>Gly</td><td>thr</td><td>thr</td><td>CYS</td><td>tyr</td><td>Ala</td><td>will</td><td>Gly</td><td>trp</td><td>Gly</td><td>will</td><td>Ile</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td>Lys</td><td>Lys</td><td>Leu</td><td>gin</td><td>CYS</td><td>Val</td><td>asp</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>val</td><td>Ile</td><td>will</td><td>asn</td><td>asp</td><td>val</td><td>CYS</td><td>Ala</td><td>gin</td><td>val</td><td>It's</td><td>pro</td><td>gin</td><td>Lys</td><td>val</td>

180

185

190

Trp Gly 225

Lys thr

CYS will 210

<td>Phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td rowspan="2">Arg</td><td>trp</td><td>thr</td><td>Gly</td><td>Gly</td><td>Lys</td><td>will</td><td>thr</td>

<td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Val</td><td>will</td><td>His</td><td>pro</td><td>tyr</td><td>will</td><td>gin</td><td rowspan="2">asp</td><td>Leu</td><td>glu</td><td rowspan="2">Gly</td><td>Lys</td><td>Gly</td><td>glu</td>

<td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>pro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 38 &lt;211&gt; 104 &lt;212&gt; PRT

-103 &lt;213&gt; Homo sapiens

<td colspan="5"> &lt;400&gt; 38</td><td rowspan="2">val</td><td colspan="3" rowspan="2">Phe Leu Thr</td><td rowspan="2">Leu 10</td><td rowspan="2">will</td><td rowspan="2">val</td><td rowspan="2">thr</td><td rowspan="2">trp</td><td rowspan="2">ile 15</td><td rowspan="2">Gly</td>

<td colspan="2">Met TRP 1</td><td>val</td><td>pro</td><td>val 5</td>

<td>glu</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>His</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>asp</td><td>Ala</td><td>Gly</td><td>glu</td><td>Gly</td><td>Ala</td><td>will</td><td>pro</td><td>asp</td>

<td></td><td> 20</td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td rowspan="2">CYS</td><td>gin</td><td>Ala</td><td>and Gl</td><td>Ala</td><td>Leu</td><td>will</td><td>pro</td><td>pro</td><td>thr</td><td>gin</td><td>His</td><td>pro</td><td>will</td><td>pro</td><td>asp</td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td rowspan="2">Arg</td><td>glu</td><td>Leu</td><td rowspan="2">Gly</td><td>will</td><td>Phe</td><td>Leu</td><td>will</td><td>Leu</td><td>pro</td><td>for a</td><td>pro</td><td>Leu</td><td>gin</td><td>Ala</td><td>His</td>

<td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>thr</td><td>pro</td><td>will</td><td>pro</td><td>will</td><td>ile</td><td>Leu</td><td>gin</td><td>gin</td><td>will</td><td>will</td><td>Leu</td><td>pro</td><td>His</td><td>gin</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>pro</td><td>Ala</td><td>pro</td><td>will</td><td>His</td><td>Leu</td><td>pro</td><td>gin</td><td>asn</td><td>Phe</td><td>Leu</td><td>pro</td><td>ile</td><td>Ala</td><td>gin</td><td>pro</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ala</td><td>pro</td><td>CYS</td><td>will</td><td>gin</td><td>Leu</td><td>Leu</td><td>tyr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

100 &lt;210&gt; 39 &lt;211&gt; 261 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 39

<td>Met</td><td rowspan="2">trp</td><td>val</td><td>pro</td><td>val</td><td>val</td><td>Phe</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Val</td><td>thr</td><td rowspan="2">trp</td><td>Ile</td><td>Gl</td><td rowspan="2">y</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Ala</td><td>Ala</td><td>pro</td><td>Leu</td><td>Ile</td><td>Leu</td><td>will</td><td rowspan="2">Arg</td><td>Ile</td><td>val</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td>glu</td><td rowspan="2">CYS</td><td>Gl</td><td>and</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td> 30</td><td></td><td></td>

<td rowspan="2">Lys</td><td>His</td><td>will</td><td>gin</td><td>pro</td><td rowspan="2">trp</td><td>gin</td><td>val</td><td>Leu</td><td>val</td><td>Ala</td><td>will</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Al</td><td>a</td>

<td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

-104-

<td>val</td><td colspan="3">CYS Gly Gly 50</td><td>val</td><td colspan="2">Val Leu 55</td><td colspan="5">His Gin Pro TRP val 60</td><td>Leu</td><td>thr</td><td>Ala</td><td>Ala</td>

<td>It's</td><td>CYS</td><td>Ile</td><td rowspan="2">Arg</td><td>asn</td><td>Lys</td><td>will</td><td>Val</td><td>Ile</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>His</td><td>will</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>phe</td><td>It's</td><td>pro</td><td>glu</td><td>asp</td><td>thr</td><td rowspan="2">Gly</td><td>gin</td><td>val</td><td>Phe</td><td>gin</td><td>val</td><td>will</td><td>His</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>pro</td><td>His</td><td>pro</td><td>Leu</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Met</td><td>will</td><td>Leu</td><td>Leu</td><td rowspan="2">Lys</td><td>asn</td><td rowspan="2">Arg</td><td>Phe</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td>¹¹⁰</td><td></td>

<td>pro</td><td rowspan="2">Gly</td><td>asp</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>His</td><td>asp</td><td>Leu</td><td>Met</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Leu</td><td>will</td><td>glu</td>

<td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>pro</td><td>Ala</td><td>glu</td><td>Leu</td><td>thr</td><td rowspan="2">asp</td><td>Ala</td><td>val</td><td rowspan="2">Lys</td><td>val</td><td>Met</td><td>asp</td><td>Leu</td><td>pro</td><td>thr</td><td>gin</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>glu</td><td>pro</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>thr</td><td>thr</td><td rowspan="2">CYS</td><td rowspan="2">tyr</td><td>Ala</td><td>will</td><td rowspan="2">Gly</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>will</td><td>ile</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td> 155</td><td></td><td> 160</td>

<td>glu</td><td>pro</td><td>glu</td><td>glu</td><td>Phe</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Lys</td><td>Lys</td><td>Leu</td><td>gin</td><td rowspan="2">CYS</td><td>val</td><td>asp</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>val</td><td>ile</td><td>will</td><td>asn</td><td rowspan="2">asp</td><td>Val</td><td rowspan="2">CYS</td><td>Ala</td><td>gin</td><td>Val</td><td>His</td><td>pro</td><td>gin</td><td rowspan="2">Lys</td><td>val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td>

<td>thr</td><td>Lys</td><td>phe</td><td>Met</td><td>Leu</td><td>CYS</td><td>Ala</td><td>Gly</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>thr</td><td rowspan="2">Gly</td><td>Gly</td><td>Lys</td><td>will</td><td>thr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td> 205</td><td></td><td></td><td></td>

<td>CYS</td><td>will</td><td>Gly</td><td>asp</td><td>will</td><td rowspan="2">Gly</td><td>Gly</td><td>pro</td><td>Leu</td><td>val</td><td rowspan="2">CYS</td><td>asn</td><td rowspan="2">Gly</td><td>Val</td><td>Leu</td><td>gin</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Gly</td><td>ile</td><td>thr</td><td>will</td><td rowspan="2">trp</td><td>Gly</td><td>will</td><td>glu</td><td>pro</td><td rowspan="2">CYS</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td rowspan="2">Arg</td><td>pro</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td>Leu</td><td>tyr</td><td>thr</td><td>Lys</td><td>val</td><td>val</td><td>His</td><td rowspan="2">tyr</td><td>Arg</td><td rowspan="2">Lys</td><td rowspan="2">trp</td><td>Ile</td><td>Lys</td><td>asp</td><td>thr</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td> 255</td><td></td>

ile val ASN Pro 260 Ala &lt;210&gt; 40 &lt;211&gt; 1729 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 40

-105aagtttccct tctcccagtc caagacccca aatcaccaca aaggacccaa tccccagact 60 caagatatgg tctgggcgct gtcttgtgtc tcctaccctg atccctgggt tcaactctgc 120 tcccagagca tgaagcctct ccaccagcac cagccaccaa cctgcaaacc tagggaagat 180 tgacagaatt cccagccttt cccagctccc cctgcccatg tcccaggact cccagccttg 240 gttctctgcc cccgtgtctt ttcaaaccca catcctaaat ccatctccta tccgagtccc 300 ccagttcctc ctgtcaaccc tgattcccct gatctagcac cccctctgca ggtgctgcac 360 ccctcatcct gtctcggatt gtgggaggct gggagtgcga gaagcattcc caaccctggc 420 aggtgcttgt agcctctcgt ggcagggcag tctgcggcgg tgttctggtg cacccccagt 480 gggtcctcac agctacccac tgcatcagga acaaaagcgt gatcttgctg ggtcggcaca 540 gcctgtttca tcctgaagac acaggccagg tatttcaggt cagccacagc ttcccacacc 600 cgctctacga tatgagcctc ctgaagaatc gattcctcag gccaggtgat gactccagcc 660 acgacctcat gctgctccgc ctgtcagagc ctgccgagct cacggatgct atgaaggtca 720 tggacctgcc cacccaggag ccagcactgg ggaccacctg ctacgcctca ggctggggca 780 gcattgaacc agaggagttc ttgaccccaa agaaacttca gtgtgtggac ctccatgtta 840 tttccaatga cgtgtgtgcg caagttcacc ctcagaaggt gaccaagttc atgctgtgtg 900 ctggacgctg gacagggggc aaaagcacct gctcgggtga ttctgggggc ccacttgtct 960 gtaatggtgt gcttcaaggt atcacgtcat ggggcagtga accatgtgcc ctgcccgaaa 1020 ggccttccct gtacaccaag gtggtgcatt accggaagtg gatcaaggac accatcgtgg 1080 ccaacccctg agcaccccta tcaactccct attgtagtaa acttggaacc ttggaaatga 1140 ccaggccaag actcaggcct ccccagttct actgaccttt gtccttaggt gtgaggtcca 1200 gggttgctag gaaaagaaat cagcagacac aggtgtagac cagagtgttt cttaaatggt 1260 gtaattttgt cctctctgtg tcctggggaa tactggccat gcctggagac atatcactca 1320 atttctctga ggacacagat aggatggggt gtctgtgtta tttgtggggt acagagatga 1380 aagaggggtg ggatccacac tgagagagtg gagagtgaca tgtgctggac actgtccatg 1440 aagcactgag cagaagctgg aggcacaacg caccagacac tcacagcaag gatggagctg 1500 aaaacataac ccactctgtc ctggaggcac tgggaagcct agagaaggct gtgaaccaag 1560 gagggagggt cttcctttgg catgggatgg ggatgaagta aggagaggga ctgaccccct 1620 ggaagctgat tcactatggg gggaggtgta ttgaagtcct ccagacaacc ctcagatttg 1680 atgatttcct agtagaactc acagaaataa agagctgtta tactgtgaa 1729 &lt;210&gt; 41 &lt;211&gt; 719 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

-106&lt;400&gt; 41

TRP Met Leu Leu Glu Thr His ASN will Ala Asp Ala Thr Arg Ala val 15 10 15

Pro Arg Leu Arg TRP CYS Ala Gly Ala Gly Ala Leu Leu Gly Phe val 20 25 30

-107-

<td>Phe</td><td colspan="5">Leu Leu Leu Gly Phe 35</td><td>Phe</td><td>Gly 40</td><td colspan="4">Trp ile Phe Lys</td><td>will 45</td><td>will</td><td>asn</td><td>glu</td>

<td>Ala</td><td>thr</td><td>asn</td><td>ile</td><td>thr</td><td>pro</td><td>Lys</td><td>His</td><td>asn</td><td>Met</td><td rowspan="2">Lys</td><td>Ala</td><td>Phe</td><td>Leu</td><td rowspan="2">asp</td><td>glu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>glu</td><td>asn</td><td>ile</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Phe</td><td>Leu</td><td>tyr</td><td>asn</td><td>Phe</td><td>thr</td><td>gin</td><td>ile</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>pro</td><td>His</td><td>Leu</td><td>Ala</td><td>Gly</td><td>thr</td><td>glu</td><td>gin</td><td>asn</td><td>Phe</td><td>gin</td><td>Leu</td><td>Ala</td><td rowspan="2">Lys</td><td>gin</td><td>Ile</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>gin</td><td>will</td><td>gin</td><td>trp</td><td>Lys</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td rowspan="2">ASP</td><td>will</td><td>val</td><td>glu</td><td>Leu</td><td>Ala</td><td>It's</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td rowspan="2">tyr</td><td>asp</td><td>val</td><td>Leu</td><td>Leu</td><td>will</td><td rowspan="2">tyr</td><td>pro</td><td>asn</td><td rowspan="2">Lys</td><td>thr</td><td>It's</td><td>pro</td><td>asn</td><td rowspan="2">tyr</td><td>Ile</td>

<td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>will</td><td>Ile</td><td>He</td><td>asn</td><td>glu</td><td rowspan="2">asp</td><td>Gly</td><td>asn</td><td>glu</td><td>ile</td><td>Phe</td><td>asn</td><td>thr</td><td>will</td><td>Leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>glu</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>Gly</td><td rowspan="2">tyr</td><td>glu</td><td>asn</td><td>Val</td><td>will</td><td rowspan="2">asp</td><td>Ile</td><td>Val</td><td>pro</td><td>pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>will</td><td>Ala</td><td>Phe</td><td>will</td><td>pro</td><td>n gl</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td>glu</td><td rowspan="2">Gly</td><td rowspan="2">asp</td><td>Leu</td><td>Val</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td> 175</td>

<td>Val</td><td>asn</td><td>tyr</td><td>Ala</td><td>Arg</td><td>thr</td><td>glu</td><td>asp</td><td>Phe</td><td>Phe</td><td>Lys</td><td>Leu</td><td>glu</td><td>Arg</td><td>asp</td><td>Met</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Lys</td><td>Ile</td><td>asn</td><td>CYS</td><td>will</td><td>Gly</td><td>Lys</td><td>Ile</td><td>val</td><td>Ile</td><td>Ala</td><td>Arg</td><td>tyr</td><td>Gly</td><td>Lys</td><td>val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Phe</td><td>Arg</td><td>Gly</td><td>asn</td><td>Lys</td><td>Val</td><td>Lys</td><td>asn</td><td>Ala</td><td>gin</td><td>Leu</td><td>Ala</td><td rowspan="2">Gly</td><td>Ala</td><td rowspan="2">Lys</td><td rowspan="2">Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td>

<td>Val</td><td>ile</td><td>Leu</td><td>tyr</td><td>will</td><td>asp</td><td>pro</td><td>Ala</td><td rowspan="2">asp</td><td rowspan="2">tyr</td><td>phe</td><td>Ala</td><td>pro</td><td rowspan="2">Gly</td><td>val</td><td>Lys</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td>tyr</td><td>pro</td><td>asp</td><td>Gly</td><td>trp</td><td>asn</td><td>Leu</td><td>pro</td><td>Gly</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>val</td><td>n gl</td><td>Arg</td><td rowspan="2">Gly</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td> 255</td>

<td>asn</td><td>Ile</td><td>Leu</td><td>asn</td><td>Leu</td><td>asn</td><td rowspan="2">Gly</td><td>Ala</td><td>Gly</td><td rowspan="2">ASP</td><td>pro</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>pro</td><td>Ala</td><td>asn</td><td>glu</td><td rowspan="2">tyr</td><td>Ala</td><td rowspan="2">tyr</td><td>Arg</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>Ile</td><td>Ala</td><td>glu</td><td>Ala</td><td>val</td><td rowspan="2">Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td> 280</td><td></td><td></td><td> 285</td><td></td><td></td>

<td>Leu</td><td>pro</td><td>will</td><td>Ile</td><td>pro</td><td>val</td><td>It's</td><td>pro</td><td>Ile</td><td>Gly</td><td>tyr</td><td>tyr</td><td>asp</td><td>Ala</td><td>gin</td><td>Lys</td>

290 295 300

-108-

<td colspan="5">Met Leu Leu Glu Lys 305</td><td>Gly 310</td><td colspan="3">Will Ala Gly</td><td colspan="3">Asp Pro Pro 315</td><td>will</td><td colspan="2">will TRP</td><td>Arg 320</td>

<td rowspan="2">Gly</td><td>will</td><td>Leu</td><td rowspan="2">Lys</td><td>Val</td><td>pro</td><td rowspan="2">tyr</td><td>asn</td><td>val</td><td>Gly</td><td>pro</td><td rowspan="2">Gly</td><td>Phe</td><td>thr</td><td>Gly</td><td>asn</td>

<td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Phe</td><td>will</td><td>thr</td><td>gin</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Met</td><td>It's</td><td>ile</td><td>It's</td><td>will</td><td>thr</td><td>asn</td><td>glu</td><td>val</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>thr</td><td>Arg</td><td>ile</td><td>tyr</td><td>asn</td><td>val</td><td>ile</td><td>Gly</td><td>thr</td><td>Leu</td><td rowspan="2">Arg</td><td>Gly</td><td>Ala</td><td>Val</td><td>glu</td><td>pro</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>asp</td><td>Arg</td><td>tyr</td><td>val</td><td>ile</td><td>Leu</td><td>Gly</td><td rowspan="2">Gly</td><td>His</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td>trp</td><td>val</td><td>Phe</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ile</td><td rowspan="2">asp</td><td>pro</td><td>gin</td><td>will</td><td rowspan="2">Gly</td><td>Ala</td><td>Ala</td><td>val</td><td>val</td><td>His</td><td>glu</td><td>Ile</td><td>val</td><td>Arg</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>will</td><td>Phe</td><td>Gly</td><td>thr</td><td>Leu</td><td>Lys</td><td>Lys</td><td>glu</td><td>Gly</td><td>trp</td><td>Arg</td><td>pro</td><td>Arg</td><td>Arg</td><td>thr</td><td>ile</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Leu</td><td>Phe</td><td>Ala</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">asp</td><td>Ala</td><td>glu</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td>Leu</td><td>Gly</td><td>will</td><td>thr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>glu</td><td rowspan="2">trp</td><td>Ala</td><td>glu</td><td>glu</td><td>asn</td><td>will</td><td>Arg</td><td>Leu</td><td>Leu</td><td>gin</td><td>and Gl</td><td>Arg</td><td rowspan="2">Gly</td><td>val</td><td>Ala</td>

<td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td>

<td rowspan="2">tyr</td><td>Ile</td><td>asn</td><td>Ala</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>ile</td><td>glu</td><td rowspan="2">Gly</td><td>asn</td><td>tyr</td><td>thr</td><td>Leu</td><td>Arg</td><td>val</td>

<td> 450</td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>asp</td><td rowspan="2">CYS</td><td>thr</td><td>pro</td><td>Leu</td><td>Met</td><td rowspan="2">tyr</td><td>will</td><td>Leu</td><td>val</td><td>It's</td><td>asn</td><td>Leu</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td>

<td> 465</td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td> 480</td>

<td>Leu</td><td rowspan="2">Lys</td><td>will</td><td>pro</td><td>asp</td><td>glu</td><td rowspan="2">Gly</td><td>Phe</td><td>glu</td><td>Gly</td><td>Lys</td><td>will</td><td>Leu</td><td>tyr</td><td>glu</td><td>will</td>

<td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td rowspan="2">trp</td><td>thr</td><td rowspan="2">Lys</td><td>Lys</td><td>will</td><td>pro</td><td>will</td><td>pro</td><td>glu</td><td>Phe</td><td>will</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td rowspan="2">Arg</td><td>ile</td>

<td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td> 510</td><td></td>

<td>will</td><td rowspan="2">Lys</td><td>Leu</td><td rowspan="2">Gly</td><td>will</td><td rowspan="2">Gly</td><td>asn</td><td>asp</td><td>Phe</td><td>glu</td><td>val</td><td>Phe</td><td>Phe</td><td>gin</td><td rowspan="2">Arg</td><td>Leu</td>

<td></td><td> 515</td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td>

<td>Gly</td><td>ile</td><td>Ala</td><td>will</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>Ala</td><td rowspan="2">Arg</td><td rowspan="2">tyr</td><td>thr</td><td>Lys</td><td>asn</td><td>trp</td><td>glu</td><td>thr</td><td>asn</td>

<td></td><td> 530</td><td></td><td></td><td> 535</td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Phe</td><td>will</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td>pro</td><td>Leu</td><td rowspan="2">tyr</td><td>His</td><td>will</td><td>val</td><td>tyr</td><td>glu</td><td>thr</td><td>tyr</td><td>glu</td>

<td> 545</td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td>

<td>Leu</td><td>val</td><td>glu</td><td rowspan="2">Lys</td><td>Phe</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>pro</td><td>Met</td><td>Phe</td><td>Lys</td><td>tyr</td><td>His</td><td>Leu</td><td>thr</td><td>val</td>

<td></td><td></td><td></td><td> 565</td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td>

-109-

<td>Ala</td><td>gin</td><td>Val</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Met</td><td>val</td><td>Phe</td><td>glu</td><td>Leu</td><td>Ala</td><td>asn</td><td>will</td><td>Ile</td><td>val</td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Leu</td><td>pro</td><td>Phe</td><td>asp</td><td>CYS</td><td rowspan="2">Arg</td><td>asp</td><td>tyr</td><td>Ala</td><td>val</td><td>val</td><td>Leu</td><td>Arg</td><td>Lys</td><td>tyr</td><td>Ala</td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td>

<td rowspan="2">asp</td><td>Lys</td><td>ile</td><td rowspan="2">tyr</td><td>will</td><td>Ile</td><td>will</td><td>Met</td><td rowspan="2">Lys</td><td>It's</td><td>pro</td><td>gin</td><td>glu</td><td>Met</td><td rowspan="2">Lys</td><td>thr</td>

<td> 610</td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td>

<td>tyr</td><td>will</td><td>val</td><td>will</td><td>Phe</td><td>asp</td><td>will</td><td>Leu</td><td>Phe</td><td>will</td><td>Ala</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>Phe</td><td>thr</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td> 640</td>

<td>glu</td><td>ile</td><td>Ala</td><td>will</td><td>Lys</td><td>Phe</td><td>will</td><td>glu</td><td rowspan="2">Arg</td><td>Leu</td><td>gin</td><td>asp</td><td>Phe</td><td>asp</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td>Lys</td><td>hl s</td><td>val</td><td>ile</td><td rowspan="2">tyr</td><td>Ala</td><td>pro</td><td>will</td><td>will</td><td>It's</td><td>asn</td><td rowspan="2">Lys</td><td rowspan="2">tyr</td><td>Ala</td><td rowspan="2">Gly</td><td>glu</td>

<td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td> 670</td><td></td>

<td>will</td><td>Phe</td><td>pro</td><td>Gly</td><td>Ile</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Ala</td><td>Leu</td><td>Phe</td><td rowspan="2">asp</td><td>ile</td><td>glu</td><td>will</td><td rowspan="2">Lys</td><td>Val</td>

<td></td><td></td><td> 675</td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td> 685</td><td></td><td></td>

<td>asp</td><td>pro</td><td>will</td><td>Lys</td><td>Ala</td><td>trp</td><td>Gly</td><td>glu</td><td>Val</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td>gin</td><td>Ile</td><td rowspan="2">tyr</td><td>val</td><td>Ala</td>

<td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td> 700</td><td></td><td></td><td></td>

<td>Ala</td><td>Phe</td><td>thr</td><td>val</td><td>gin</td><td>Ala</td><td>Ala</td><td>Ala</td><td>glu</td><td>thr</td><td>Leu</td><td>will</td><td>glu</td><td>val</td><td>Ala</td><td></td>

<td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 42 &lt;211&gt; 2472 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 42

-110-

<td>ctggacccca</td><td>ggtctggagc</td><td>gaattccagc</td><td>ctgcagggct</td><td>gataagcgag</td><td>gcattagtga</td><td> 60</td>

<td>gattgagaga</td><td>gactttaccc</td><td>cgccgtggtg</td><td>gttggagggc</td><td>gcgcagtaga</td><td>gcagcagcac</td><td> 120</td>

<td>aggcgcgggt</td><td>cccgggaggc</td><td>cggctctgct</td><td>cgcgccgaga</td><td>tgtggaatct</td><td>ccttcacgaa</td><td> 180</td>

<td>accgactcgg</td><td>ctgtggccac</td><td>cgcgcgccgc</td><td>ccgcgctggc</td><td>tgtgcgctgg</td><td>ggcgctggtg</td><td> 240</td>

<td>ctggcgggtg</td><td>gettetttet</td><td>cctcggcttc</td><td>ctcttcgggt</td><td>ggtttataaa</td><td>atcctccaat</td><td> 300</td>

<td>gaagctacta</td><td>acattactcc</td><td>aaagcataat</td><td>atgaaagcat</td><td>ttttggatga</td><td>attgaaagct</td><td> 360</td>

<td>gagaacatca</td><td>agaagttctt</td><td>atataatttt</td><td>acacagatac</td><td>cacatttagc</td><td>aggaacagaa</td><td> 420</td>

<td>caaaactttc</td><td>agcttgcaaa</td><td>gcaaattcaa</td><td>tcccagtgga</td><td>aagaatttgg</td><td>cctggattct</td><td> 480</td>

<td>gttgagetag</td><td>cacattatga</td><td>tgtcctgttg</td><td>tcctacccaa</td><td>ataagactca</td><td>tcccaactac</td><td> 540</td>

<td>atctcaataa</td><td>ttaatgaaga</td><td>tggaaatgag</td><td>attttcaaca</td><td>catcattatt</td><td>tgaaccacct</td><td> 600</td>

<td>cctccaggat</td><td>atgaaaatgt</td><td>tteggatatt</td><td>gtaccacctt</td><td>tcagtgcttt</td><td>ctctcctcaa</td><td> 660</td>

<td>ggaatgccag</td><td>agggcgatct</td><td>agtgtatgtt</td><td>aactatgcac</td><td>gaactgaaga</td><td>cttctttaaa</td><td> 720</td>

<td>ttggaacggg</td><td>acatgaaaat</td><td>caattgctct</td><td>gggaaaattg</td><td>taattgccag</td><td>atatgggaaa</td><td> 780</td>

-111-

<td>gttttcagag</td><td>gaaataaggt</td><td>taaaaatgcc</td><td>cagctggcag</td><td>gggccaaagg</td><td>agtcattctc</td><td> 840</td>

<td>tactccgacc</td><td>ctgctgacta</td><td>ctttgctcct</td><td>ggggtgaagt</td><td>cctatccaga</td><td>tggttggaat</td><td> 900</td>

<td>cttcctggag</td><td>gtggtgtcca</td><td>gcgtggaaat</td><td>atcctaaatc</td><td>tgaatggtgc</td><td>aggagaccct</td><td> 960</td>

<td>ctcacaccag</td><td>gttacccagc</td><td>aaatgaatat</td><td>gcttataggc</td><td>gtggaattgc</td><td>agaggctgtt</td><td> 1020</td>

<td>ggtcttccaa</td><td>gtattcctgt</td><td>tcatccaatt</td><td>ggatactatg</td><td>atgcacagaa</td><td>gctcctagaa</td><td> 1080</td>

<td>aaaatgggtg</td><td>gctcagcacc</td><td>accagatagc</td><td>agctggagag</td><td>gaagtctcaa</td><td>agtgccctac</td><td> 1140</td>

<td>aatgttggac</td><td>ctggctttac</td><td>tggaaacttt</td><td>tctacacaaa</td><td>aagtcaagat</td><td>gcacatccac</td><td> 1200</td>

<td>tctaccaatg</td><td>aagtgacaag</td><td>aatttacaat</td><td>gtgataggta</td><td>ctctcagagg</td><td>agcagtggaa</td><td> 1260</td>

<td>ccagacagat</td><td>atgtcattct</td><td>gggaggtcac</td><td>cgggactcat</td><td>gggtgtttgg</td><td>tggtattgac</td><td> 1320</td>

<td>cctcagagtg</td><td>gagcagctgt</td><td>tgttcatgaa</td><td>attgtgagga</td><td>gctttggaac</td><td>actgaaaaag</td><td> 1380</td>

<td>gaagggtgga</td><td>gacctagaag</td><td>aacaattttg</td><td>tttgcaagct</td><td>gggatgcaga</td><td>agaatttggt</td><td> 1440</td>

<td>cttcttggtt</td><td>ctactgagtg</td><td>ggcagaggag</td><td>aattcaagac</td><td>tccttcaaga</td><td>gcgtggcgtg</td><td> 1500</td>

<td>gcttatatta</td><td>atgctgactc</td><td>atctatagaa</td><td>ggaaactaca</td><td>ctctgagagt</td><td>tgattgtaca</td><td> 1560</td>

<td>ccgctgatgt</td><td>acagcttggt</td><td>acacaaccta</td><td>acaaaagagc</td><td>tgaaaagccc</td><td>tgatgaaggc</td><td> 1620</td>

<td>tttgaaggca</td><td>aatctcttta</td><td>tgaaagttgg</td><td>actaaaaaaa</td><td>gtccttcccc</td><td>agagttcagt</td><td> 1680</td>

<td>ggcatgccca</td><td>ggataagcaa</td><td>attgggatct</td><td>ggaaatgatt</td><td>ttgaggtgtt</td><td>cttccaacga</td><td> 1740</td>

<td>cttggaattg</td><td>cttcaggcag</td><td>agcacggtat</td><td>actaaaaatt</td><td>gggaaacaaa</td><td>caaattcagc</td><td> 1800</td>

<td>ggctatccac</td><td>tgtatcacag</td><td>tgtctatgaa</td><td>acatatgagt</td><td>tggtggaaaa</td><td>gttttatgat</td><td> 1860</td>

<td>ccaatgttta</td><td>aatatcacct</td><td>cactgtggcc</td><td>caggttcgag</td><td>gagggatggt</td><td>gtttgagcta</td><td> 1920</td>

<td>gccaattcca</td><td>tagtgctccc</td><td>ttttgattgt</td><td>cgagattatg</td><td>ctgtagtttt</td><td>aagaaagtat</td><td> 1980</td>

<td>gctgacaaaa</td><td>tctacagtat</td><td>ttctatgaaa</td><td>catccacagg</td><td>aaatgaagac</td><td>atacagtgta</td><td> 2040</td>

<td>tcatttgatt</td><td>cacttttttc</td><td>tgcagtaaag</td><td>aattttacag</td><td>aaattgcttc</td><td>caagttcagt</td><td> 2100</td>

<td>gagagactcc</td><td>aggactttga</td><td>caaaagcaag</td><td>catgtcatct</td><td>atgctccaag</td><td>cagccacaac</td><td> 2160</td>

<td>aagtatgcag</td><td>gggagtcatt</td><td>cccaggaatt</td><td>tatgatgctc</td><td>tgtttgatat</td><td>tgaaagcaaa</td><td> 2220</td>

<td>gtggaccctt</td><td>ccaaggcctg</td><td>gggagaagtg</td><td>aagagacaga</td><td>tttatgttgc</td><td>agccttcaca</td><td> 2280</td>

<td>gtgcaggcag</td><td>ctgcagagac</td><td>tttgagtgaa</td><td>gtagcctaag</td><td>aggattcttt</td><td>agagaatccg</td><td> 2340</td>

<td>tattgaattt</td><td>gtgtggtatg</td><td>tcactcagaa</td><td>agaatcgtaa</td><td>tgggtatatt</td><td>gataaatttt</td><td> 2400</td>

<td>aaaattggta</td><td>tatttgaaat</td><td>aaagttgaat</td><td>attatatata</td><td>aaaaaaaaaa</td><td>aaaaaaaaaa</td><td> 2460</td>

<td>aaaaaaaaaa</td><td>aa</td><td></td><td></td><td></td><td></td><td> 2472</td>

&lt;210&gt; 43 &lt;211&gt; 719 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

-112&lt;400&gt; 43

TRP Met Leu Leu Glu Thr His ASN will Ala Asp Ala Thr Arg Ala val

-113-

<td colspan="3" rowspan="2">1 Arg Pro Arg</td><td rowspan="2">trp 20</td><td colspan="4"> 5</td><td colspan="3"> 10 '</td><td colspan="2" rowspan="2">Leu Ala</td><td colspan="3"> 15</td>

<td colspan="3">Leu Ala CYS</td><td>Gly</td><td>Ala 25</td><td>Leu</td><td>val</td><td>Gly 30</td><td>Gly</td><td>Phe</td>

<td>Phe</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Gly</td><td rowspan="2">trp</td><td>Phe</td><td>Ile</td><td rowspan="2">Lys</td><td>will</td><td>will</td><td>asn</td><td>glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ala</td><td>thr</td><td>asn</td><td>Ile</td><td>thr</td><td>pro</td><td>Lys</td><td>His</td><td>asn</td><td>Met</td><td rowspan="2">Lys</td><td>Ala</td><td>Phe</td><td>Leu</td><td rowspan="2">asp</td><td>glu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>glu</td><td>asn</td><td>Ile</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Phe</td><td>Leu</td><td>tyr</td><td>asn</td><td>Phe</td><td>thr</td><td>gin</td><td>Ile</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>pro</td><td>His</td><td>Leu</td><td>Ala</td><td>Gly</td><td>thr</td><td>glu</td><td>gin</td><td>asn</td><td>Phe</td><td>gin</td><td>Leu</td><td>Ala</td><td rowspan="2">Lys</td><td>gin</td><td>ile</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>gin</td><td>will</td><td>gin</td><td>trp</td><td rowspan="2">Lys</td><td>glu</td><td>phe</td><td rowspan="2">Gly</td><td>Leu</td><td rowspan="2">asp</td><td>will</td><td>Val</td><td>glu</td><td>Leu</td><td>Ala</td><td>His</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Val</td><td>Leu</td><td>Leu</td><td>will</td><td rowspan="2">tyr</td><td>pro</td><td>asn</td><td rowspan="2">Lys</td><td>thr</td><td>It's</td><td>pro</td><td>asn</td><td rowspan="2">tyr</td><td>ile</td>

<td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>will</td><td>ile</td><td>ile</td><td>asn</td><td>glu</td><td rowspan="2">asp</td><td>Gly</td><td>asn</td><td>glu</td><td>ile</td><td>Phe</td><td>asn</td><td>thr</td><td>will</td><td>Leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>glu</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>Gly</td><td rowspan="2">tyr</td><td>glu</td><td>asn</td><td>val</td><td>will</td><td rowspan="2">asp</td><td>ile</td><td>val</td><td>pro</td><td>pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>will</td><td>Ala</td><td>Phe</td><td>will</td><td>pro</td><td>gin</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td>glu</td><td rowspan="2">Gly</td><td rowspan="2">asp</td><td>Leu</td><td>val</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td> 175</td>

<td>val</td><td>asn</td><td>tyr</td><td>Ala</td><td rowspan="2">Arg</td><td>thr</td><td>glu</td><td>asp</td><td>Phe</td><td>Phe</td><td>Lys</td><td>Leu</td><td>glu</td><td>Arg</td><td>ASP</td><td>Met</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Lys</td><td>ile</td><td>asn</td><td>CYS</td><td>will</td><td>Gly</td><td rowspan="2">Lys</td><td>Ile</td><td>val</td><td>ile</td><td>Ala</td><td rowspan="2">Arg</td><td>tyr</td><td>Gly</td><td rowspan="2">Lys</td><td>val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>Phe</td><td>Arg</td><td>Gly</td><td>asn</td><td>Lys</td><td>val</td><td>Lys</td><td>asn</td><td>Ala</td><td>gin</td><td>Leu</td><td>Ala</td><td rowspan="2">Gly</td><td>Ala</td><td>Lys</td><td>Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>val</td><td>ile</td><td>Leu</td><td>tyr</td><td>will</td><td>asp</td><td>pro</td><td>Ala</td><td>asp</td><td>tyr</td><td>Phe</td><td>Ala</td><td>pro</td><td rowspan="2">Gly</td><td>val</td><td>Lys</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td rowspan="2">tyr</td><td>pro</td><td rowspan="2">asp</td><td>Gly</td><td>trp</td><td>asn</td><td>Leu</td><td>pro</td><td>Gly</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Val</td><td>gin</td><td>Arg</td><td rowspan="2">Gly</td>

<td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td> 255</td>

<td>asn</td><td>Ile</td><td>Leu</td><td>asn</td><td>Leu</td><td>asn</td><td rowspan="2">Gly</td><td>Ala</td><td>Gly</td><td rowspan="2">asp</td><td>pro</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>pro</td><td>Ala</td><td>asn</td><td>glu</td><td>tyr</td><td>Ala</td><td>tyr</td><td>Arg</td><td>Arg</td><td>Gly</td><td>Ile</td><td>Ala</td><td>glu</td><td>Ala</td><td>Val</td><td>Gly</td>

-114-

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Leu</td><td>pro</td><td>will</td><td>Ile</td><td>pro</td><td>val</td><td>His</td><td>pro</td><td>Ile</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td>tyr</td><td rowspan="2">asp</td><td>Ala</td><td>gin</td><td>Lys</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>glu</td><td rowspan="2">Lys</td><td>Met</td><td>Gly</td><td rowspan="2">Gly</td><td>will</td><td>Ala</td><td>pro</td><td>pro</td><td rowspan="2">ASP</td><td>will</td><td>will</td><td>trp</td><td>Arg</td>

<td> 305</td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td> 320</td>

<td rowspan="2">Gly</td><td>will</td><td>Leu</td><td rowspan="2">Lys</td><td>val</td><td>pro</td><td rowspan="2">tyr</td><td>asn</td><td>val</td><td>Gly</td><td>pro</td><td rowspan="2">Gly</td><td>Phe</td><td>thr</td><td>Gly</td><td>asn</td>

<td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Phe</td><td>will</td><td>thr</td><td>gin</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Met</td><td>It's</td><td>Ile</td><td>It's</td><td>will</td><td>thr</td><td>asn</td><td>glu</td><td>val</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>thr</td><td rowspan="2">Arg</td><td>Ile</td><td rowspan="2">tyr</td><td>asn</td><td>val</td><td>Ile</td><td>Gly</td><td>thr</td><td>Leu</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>Ala</td><td>val</td><td>glu</td><td>pro</td>

<td></td><td> 355</td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>asp</td><td>Arg</td><td>tyr</td><td>Val</td><td>ile</td><td>Leu</td><td>Gly</td><td>Gly</td><td>It's</td><td rowspan="2">Arg</td><td rowspan="2">asp</td><td>will</td><td>trp</td><td>val</td><td>Phe</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ile</td><td rowspan="2">asp</td><td>pro</td><td>gin</td><td>will</td><td rowspan="2">Gly</td><td>Ala</td><td>for a</td><td>val</td><td>val</td><td>His</td><td>glu</td><td>Ile</td><td>val</td><td>Arg</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>will</td><td>Phe</td><td>Gly</td><td>thr</td><td>Leu</td><td>Lys</td><td>Lys</td><td>glu</td><td>Gly</td><td>trp</td><td>Arg</td><td>pro</td><td>Arg</td><td>Arg</td><td>thr</td><td>ile</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Leu</td><td>Phe</td><td>Ala</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">asp</td><td>Ala</td><td>glu</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td>Leu</td><td>Gly</td><td>will</td><td>thr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>glu</td><td rowspan="2">trp</td><td>Ala</td><td>glu</td><td>glu</td><td>asn</td><td>will</td><td>Arg</td><td>Leu</td><td>Leu</td><td>gin</td><td>glu</td><td>Arg</td><td rowspan="2">Gly</td><td>val</td><td>Ala</td>

<td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td>

<td>tyr</td><td>ile</td><td>asn</td><td>Ala</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>ile</td><td>glu</td><td rowspan="2">Gly</td><td>asn</td><td>tyr</td><td>thr</td><td>Leu</td><td rowspan="2">Arg</td><td>val</td>

<td></td><td> 450</td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td>

<td>asp</td><td rowspan="2">CYS</td><td>thr</td><td>pro</td><td>Leu</td><td>Met</td><td rowspan="2">tyr</td><td>will</td><td>Leu</td><td>val</td><td>It's</td><td>asn</td><td>Leu</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td>

<td> 465</td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td> 480</td>

<td>Leu</td><td>Lys</td><td>will</td><td>pro</td><td>asp</td><td>glu</td><td rowspan="2">Gly</td><td>Phe</td><td>glu</td><td>Gly</td><td rowspan="2">Lys</td><td>will</td><td>Leu</td><td rowspan="2">tyr</td><td>glu</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td> 495</td><td></td>

<td>trp</td><td>thr</td><td rowspan="2">Lys</td><td>Lys</td><td>will</td><td>pro</td><td>will</td><td>pro</td><td>glu</td><td>Phe</td><td>will</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td rowspan="2">Arg</td><td>ile</td>

<td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td> 510</td><td></td>

<td>will</td><td rowspan="2">Lys</td><td>Leu</td><td rowspan="2">Gly</td><td>will</td><td rowspan="2">Gly</td><td>asn</td><td>asp</td><td>Phe</td><td>glu</td><td>val</td><td>Phe</td><td>Phe</td><td>gin</td><td rowspan="2">Arg</td><td>Leu</td>

<td></td><td> 515</td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td>

<td>Gly</td><td>ile</td><td>Ala</td><td>will</td><td>Gly</td><td rowspan="2">Arg</td><td>Ala</td><td rowspan="2">Arg</td><td>tyr</td><td>thr</td><td>Lys</td><td>asn</td><td>trp</td><td>glu</td><td>thr</td><td>asn</td>

<td></td><td> 530</td><td></td><td></td><td></td><td>⁵³⁵</td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Lys</td><td>Phe</td><td>will</td><td>Gly</td><td>tyr</td><td>pro</td><td>Leu</td><td>tyr</td><td>His</td><td>will</td><td>val</td><td>tyr</td><td>glu</td><td>thr</td><td>tyr</td><td>glu</td>

-115-

<td colspan="2"> 545</td><td colspan="8"> 550 '</td><td rowspan="2">555 Lys</td><td rowspan="2">tyr</td><td rowspan="2">His</td><td rowspan="2">Leu</td><td rowspan="2">thr 575</td><td rowspan="2">560 val</td>

<td>Leu</td><td>val</td><td>glu</td><td>Lys</td><td colspan="3">Phe Tyr Asp 565</td><td colspan="2">pro Met</td><td>Phe 570</td>

<td>Ala</td><td>gin</td><td>Val</td><td>Arg</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Met</td><td>val</td><td>Phe</td><td>glu</td><td>Leu</td><td>Ala</td><td>asn</td><td>will</td><td>Ile</td><td>Val</td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Leu</td><td>pro</td><td>Phe</td><td>asp</td><td rowspan="2">CYS</td><td rowspan="2">Arg</td><td rowspan="2">asp</td><td>tyr</td><td>Ala</td><td>Val</td><td>Val</td><td>Leu</td><td>Arg</td><td>Lys</td><td>tyr</td><td>Ala</td>

<td></td><td></td><td> 595</td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td>

<td rowspan="2">asp</td><td>Lys</td><td>ile</td><td rowspan="2">tyr</td><td>will</td><td>Ile</td><td>will</td><td>Met</td><td rowspan="2">Lys</td><td>It's</td><td>pro</td><td>gin</td><td>glu</td><td>Met</td><td>Lys</td><td>thr</td>

<td> 610</td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td>

<td>tyr</td><td>will</td><td>val</td><td>will</td><td>Phe</td><td>asp</td><td>will</td><td>Leu</td><td>Phe</td><td>will</td><td>Ala</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>Phe</td><td>thr</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td> 640</td>

<td>glu</td><td>Ile</td><td>Ala</td><td>will</td><td>Lys</td><td>Phe</td><td>will</td><td>glu</td><td rowspan="2">Arg</td><td>Leu</td><td>gin</td><td rowspan="2">asp</td><td>Phe</td><td>asp</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td> 655</td><td></td>

<td rowspan="2">Lys</td><td>His</td><td>Val</td><td>ile</td><td rowspan="2">tyr</td><td>Ala</td><td>pro</td><td>will</td><td>will</td><td>His</td><td>asn</td><td rowspan="2">Lys</td><td>tyr</td><td>Ala</td><td>Gly</td><td>glu</td>

<td></td><td></td><td> 660</td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td>will</td><td>Phe</td><td>pro</td><td rowspan="2">Gly</td><td>Ile</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Ala</td><td>Leu</td><td>Phe</td><td rowspan="2">asp</td><td>ile</td><td>glu</td><td>will</td><td>Lys</td><td>val</td>

<td></td><td></td><td> 675</td><td></td><td> 680</td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td rowspan="2">asp</td><td>pro</td><td>will</td><td>Lys</td><td>Ala</td><td rowspan="2">trp</td><td>Gly</td><td>glu</td><td>val</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td>gin</td><td>ile</td><td>tyr</td><td>val</td><td>Ala</td>

<td> 690</td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Phe</td><td>thr</td><td>val</td><td>gin</td><td>Ala</td><td>Ala</td><td>Ala</td><td>glu</td><td>thr</td><td>Leu</td><td>will</td><td>glu</td><td>val</td><td>Ala</td><td></td>

705 710 715 &lt;210&gt; 44 &lt;211&gt; 750 &lt;212&gt; PRT

<td colspan="2">&lt;213&gt; the</td><td>Homo</td><td>SAP^-</td><td>iens</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;400&gt; </td><td> 44</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Met</td><td rowspan="2">trp</td><td>asn</td><td>Leu</td><td>Leu</td><td>His</td><td>glu</td><td>thr</td><td rowspan="2">asp</td><td>will</td><td>Ala</td><td>Val</td><td>Ala</td><td>thr</td><td>Ala</td><td rowspan="2">Arg</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td>

<td rowspan="2">Arg</td><td>pro</td><td rowspan="2">Arg</td><td>trp</td><td>Leu</td><td>CYS</td><td>Ala</td><td rowspan="2">Gly</td><td>Ala</td><td>Leu</td><td>val</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Phe</td>

<td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Phe</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Gly</td><td rowspan="2">trp</td><td>Phe</td><td>Ile</td><td rowspan="2">Lys</td><td>will</td><td>will</td><td>asn</td><td>glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ala</td><td>thr</td><td>asn</td><td>ile</td><td>thr</td><td>pro</td><td>Lys</td><td>His</td><td>asn</td><td>Met</td><td rowspan="2">Lys</td><td>Ala</td><td>Phe</td><td>Leu</td><td rowspan="2">asp</td><td>glu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>glu</td><td>asn</td><td>ile</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>Phe</td><td>Leu</td><td>tyr</td><td>asn</td><td>Phe</td><td>thr</td><td>gin</td><td>Ile</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

-116-

<td rowspan="2">pro</td><td rowspan="2">His</td><td rowspan="2">Leu</td><td rowspan="2">Ala</td><td colspan="5" rowspan="2">Thr Gly Glu Gin ASN 85</td><td colspan="6">Leu Lys Ala Phe Gin Gin</td><td rowspan="2">Ile</td>

<td colspan="4"> 90</td><td colspan="2"> 95</td>

<td>gin</td><td>will</td><td>gin</td><td>trp</td><td rowspan="2">Lys</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td rowspan="2">asp</td><td>will</td><td>val</td><td>glu</td><td>Leu</td><td>Ala</td><td>It's</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td rowspan="2">tyr</td><td rowspan="2">asp</td><td>val</td><td>Leu</td><td>Leu</td><td>will</td><td rowspan="2">tyr</td><td>pro</td><td>asn</td><td rowspan="2">Lys</td><td>thr</td><td>It's</td><td>pro</td><td>asn</td><td>tyr</td><td>ile</td>

<td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>will</td><td>Ile</td><td>Ile</td><td>asn</td><td>glu</td><td rowspan="2">asp</td><td>Gly</td><td>asn</td><td>glu</td><td>Ile</td><td>Phe</td><td>asn</td><td>thr</td><td>will</td><td>Leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>glu</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>Gly</td><td rowspan="2">tyr</td><td>glu</td><td>asn</td><td>val</td><td>will</td><td rowspan="2">asp</td><td>Ile</td><td>val</td><td>pro</td><td>pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>will</td><td>Ala</td><td>Phe</td><td>will</td><td>pro</td><td>n gl</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td>glu</td><td rowspan="2">Gly</td><td rowspan="2">asp</td><td>Leu</td><td>Val</td><td>tyr</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>asn</td><td>tyr</td><td>Ala</td><td rowspan="2">Arg</td><td>thr</td><td>glu</td><td>asp</td><td>Phe</td><td>phe</td><td>Lys</td><td>Leu</td><td>glu</td><td>Arg</td><td>asp</td><td>Met</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Lys</td><td>Ile</td><td>asn</td><td>CYS</td><td>will</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td><td>ile</td><td>val</td><td>Ile</td><td>Ala</td><td rowspan="2">Arg</td><td>tyr</td><td>Gly</td><td>Lys</td><td>val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Phe</td><td>Arg</td><td>Gly</td><td>asn</td><td>Lys</td><td>val</td><td>Lys</td><td>asn</td><td>for a</td><td>gin</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Lys</td><td>Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Ile</td><td>Leu</td><td rowspan="2">tyr</td><td>will</td><td>asp</td><td>pro</td><td>Ala</td><td rowspan="2">asp</td><td rowspan="2">tyr</td><td>Phe</td><td>Ala</td><td>pro</td><td>Gly</td><td>val</td><td>Lys</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td rowspan="2">tyr</td><td>pro</td><td>asp</td><td>Gly</td><td>trp</td><td>asn</td><td>Leu</td><td>pro</td><td>Gly</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>val</td><td>gin</td><td>Arg</td><td>Gly</td>

<td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td> 255</td><td></td>

<td>asn</td><td>Ile</td><td>Leu</td><td>asn</td><td>Leu</td><td>asn</td><td rowspan="2">Gly</td><td>Ala</td><td>Gly</td><td rowspan="2">asp</td><td>pro</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>pro</td><td>Ala</td><td>asn</td><td>glu</td><td rowspan="2">tyr</td><td>Ala</td><td>tyr</td><td>Arg</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>Ile</td><td>Ala</td><td>glu</td><td>Ala</td><td>Val</td><td>Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Leu</td><td>pro</td><td>will</td><td>ile</td><td>pro</td><td>val</td><td>It's</td><td>pro</td><td>ile</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td>tyr</td><td>asp</td><td>Ala</td><td>gin</td><td>Lys</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>glu</td><td>Lys</td><td>Met</td><td colspan="2">Gly Gly</td><td>will</td><td>Ala</td><td>pro</td><td>pro</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>trp</td><td>Arg</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td> 320</td>

<td>Gly</td><td>will</td><td>Leu</td><td>Lys</td><td>val</td><td colspan="2">pro Tyr</td><td>asn</td><td>val</td><td>Gly</td><td>pro</td><td rowspan="2">Gly</td><td>Phe</td><td>thr</td><td>Gly</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Phe</td><td>will</td><td>thr</td><td>gin</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Met</td><td>His</td><td>Ile</td><td>His</td><td>will</td><td>thr</td><td>asn</td><td>glu</td><td>val</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

-117-

<td colspan="2">thr Arg</td><td>Ile 355</td><td>tyr</td><td>asn</td><td>Val</td><td colspan="3">Ile Thr Gly 360</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Ala 365</td><td>Val</td><td>glu</td><td>pro</td>

<td>asp</td><td>Arg</td><td>tyr</td><td>val</td><td>Ile</td><td>Leu</td><td>Gly</td><td rowspan="2">Gly</td><td>His</td><td rowspan="2">Arg</td><td rowspan="2">asp</td><td>will</td><td>trp</td><td>Val</td><td>Phe</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>ile</td><td rowspan="2">asp</td><td>pro</td><td>gin</td><td>will</td><td rowspan="2">Gly</td><td>Ala</td><td>Ala</td><td>val</td><td>val</td><td>His</td><td>glu</td><td>Ile</td><td>Val</td><td>Arg</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>will</td><td>Phe</td><td>Gly</td><td>thr</td><td>Leu</td><td>Lys</td><td>Lys</td><td>glu</td><td rowspan="2">Gly</td><td>trp</td><td rowspan="2">Arg</td><td>pro</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>thr</td><td>Ile</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td> 410</td><td></td><td> 415</td><td></td>

<td>Leu</td><td>Phe</td><td>Ala</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">asp</td><td>Ala</td><td>glu</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td>Leu</td><td>Gly</td><td>will</td><td>thr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>glu</td><td>trp</td><td>Ala</td><td>glu</td><td>glu</td><td>asn</td><td>will</td><td>Arg</td><td>Leu</td><td>Leu</td><td>gin</td><td>glu</td><td>Arg</td><td rowspan="2">Gly</td><td>Val</td><td>Ala</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td>

<td>tyr</td><td>Ile</td><td>asn</td><td>Ala</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Ile</td><td>glu</td><td rowspan="2">Gly</td><td>asn</td><td>tyr</td><td>thr</td><td>Leu</td><td rowspan="2">Arg</td><td>val</td>

<td></td><td> 450</td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td>

<td>asp</td><td>CYS</td><td>thr</td><td>pro</td><td>Leu</td><td>Met</td><td rowspan="2">tyr</td><td>will</td><td>Leu</td><td>val</td><td>It's</td><td>asn</td><td>Leu</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td> 480</td>

<td>Leu</td><td>Lys</td><td>will</td><td>pro</td><td>asp</td><td>glu</td><td rowspan="2">Gly</td><td>Phe</td><td>glu</td><td>Gly</td><td rowspan="2">Lys</td><td>will</td><td>Leu</td><td rowspan="2">tyr</td><td>glu</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td> 495</td><td></td>

<td>trp</td><td>thr</td><td rowspan="2">Lys</td><td>Lys</td><td>will</td><td>pro</td><td>will</td><td>pro</td><td>glu</td><td>Phe</td><td>will</td><td rowspan="2">Gly</td><td>Met</td><td>pro</td><td rowspan="2">Arg</td><td>ile</td>

<td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td> 510</td><td></td>

<td>will</td><td rowspan="2">Lys</td><td>Leu</td><td rowspan="2">Gly</td><td>will</td><td rowspan="2">Gly</td><td>asn</td><td>asp</td><td>Phe</td><td>glu</td><td>val</td><td>Phe</td><td>Phe</td><td>gin</td><td rowspan="2">Arg</td><td>Leu</td>

<td></td><td> 515</td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td>

<td>Gly</td><td>ile</td><td>Ala</td><td>will</td><td>Gly</td><td rowspan="2">Arg</td><td>Ala</td><td rowspan="2">Arg</td><td rowspan="2">tyr</td><td>thr</td><td rowspan="2">Lys</td><td>asn</td><td rowspan="2">trp</td><td>glu</td><td>thr</td><td>asn</td>

<td></td><td> 530</td><td></td><td></td><td></td><td> 535</td><td></td><td> 540</td><td></td><td></td><td></td>

<td>Lys</td><td>Phe</td><td>will</td><td>Gly</td><td>tyr</td><td>pro</td><td>Leu</td><td rowspan="2">tyr</td><td>It's</td><td>will</td><td>val</td><td rowspan="2">tyr</td><td>glu</td><td>thr</td><td rowspan="2">tyr</td><td>glu</td>

<td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td> 560</td>

<td>Leu</td><td>Val</td><td>glu</td><td>Lys</td><td>Phe</td><td rowspan="2">tyr</td><td>asp</td><td>pro</td><td>Met</td><td>Phe</td><td>Lys</td><td rowspan="2">tyr</td><td>His</td><td>Leu</td><td>thr</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td> 575</td><td></td>

<td>Ala</td><td>gin</td><td>val</td><td>Arg</td><td colspan="2">Gly Gly</td><td>Met</td><td>val</td><td>Phe</td><td>glu</td><td>Leu</td><td>Ala</td><td>asn</td><td>will</td><td>Ile</td><td>val</td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Leu</td><td>pro</td><td>Phe</td><td>asp</td><td>CYS</td><td>Arg</td><td>asp</td><td>tyr</td><td>Ala</td><td>val</td><td>val</td><td>Leu</td><td>Arg</td><td rowspan="2">Lys</td><td rowspan="2">tyr</td><td>Ala</td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td>

<td>asp</td><td>Lys</td><td>Ile</td><td>tyr</td><td>will</td><td>ile</td><td>will</td><td>Met</td><td>Lys</td><td>His</td><td>pro</td><td>gin</td><td>glu</td><td>Met</td><td>Lys</td><td>thr</td>

610 615 620

-118-

<td>tyr 625</td><td colspan="2">Val will</td><td>will</td><td colspan="2">Phe Asp 630</td><td>will</td><td colspan="4">Will Phe Leu Ala 635</td><td>val</td><td>Lys</td><td>asn</td><td>phe</td><td>thr 640</td>

<td>glu</td><td>Ile</td><td>Ala</td><td>will</td><td>Lys</td><td>Phe</td><td>will</td><td>glu</td><td rowspan="2">Arg</td><td>Leu</td><td>gin</td><td>asp</td><td>Phe</td><td>asp</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td>asn</td><td>pro</td><td>Ile</td><td>val</td><td>Leu</td><td rowspan="2">Arg</td><td>Met</td><td>Met</td><td>asn</td><td rowspan="2">asp</td><td>gin</td><td>Leu</td><td>Met</td><td>Phe</td><td>Leu</td><td>glu</td>

<td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td>Arg</td><td>Ala</td><td>phe</td><td>Ile</td><td>asp</td><td>pro</td><td>Leu</td><td>Gly</td><td>Leu</td><td>pro</td><td>asp</td><td>Arg</td><td>pro</td><td>Phe</td><td>tyr</td><td>Arg</td>

<td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td>It's</td><td>val</td><td>Ile</td><td rowspan="2">tyr</td><td>Ala</td><td>pro</td><td>will</td><td>will</td><td>His</td><td>asn</td><td rowspan="2">Lys</td><td>tyr</td><td>Ala</td><td rowspan="2">Gly</td><td>glu</td><td>will</td>

<td></td><td> 690</td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td>

<td>Phe</td><td>pro</td><td>Gly</td><td>Ile</td><td rowspan="2">tyr</td><td>asp</td><td>Ala</td><td>Leu</td><td>Phe</td><td rowspan="2">asp</td><td>Ile</td><td>glu</td><td>will</td><td>Lys</td><td>Val</td><td>asp</td>

<td> 705</td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td>pro</td><td>will</td><td>Lys</td><td>Ala</td><td>trp</td><td>Gly</td><td>glu</td><td>val</td><td rowspan="2">Lys</td><td>Arg</td><td>gin</td><td>Ile</td><td>tyr</td><td>val</td><td>Ala</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td>

<td>Phe</td><td>thr</td><td>val</td><td>gin</td><td>Ala</td><td>Ala</td><td>Ala</td><td>glu</td><td>thr</td><td>Leu</td><td>will</td><td>glu</td><td>val</td><td>Ala</td><td></td><td></td>

<td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td><td></td><td></td>

&lt;210&gt; 45 &lt;211&gt; 671 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 45

-119-

<td>ile 1</td><td>pro</td><td>His</td><td>Leu</td><td>Ala 5</td><td>Gly</td><td>thr</td><td>glu</td>

<td>Ile</td><td>gin</td><td>will</td><td>gin 20</td><td>trp</td><td>Lys</td><td>glu</td><td>Phe</td>

<td>It's</td><td>tyr</td><td>asp 35</td><td>val</td><td>Leu</td><td>Leu</td><td>will</td><td>tyr 40</td>

<td>ile</td><td>will 50</td><td>ile</td><td>Ile</td><td>asn</td><td>glu</td><td>asp 55</td><td>Gly</td>

<td>Phe 65</td><td>glu</td><td>pro</td><td>pro</td><td>pro</td><td>pro 70</td><td>Gly</td><td>tyr</td>

<td>pro</td><td>Phe</td><td>will</td><td>Ala</td><td>Phe 85</td><td>will</td><td>pro</td><td>gin</td>

<td>tyr</td><td>val</td><td>asn</td><td>tyr 100</td><td>Ala</td><td>Arg</td><td>thr</td><td>glu</td>

ASN Phe Gin Gin Gin Leu Lys Ala 10 15

Gly Leu Leu Ala Glu Asp will val 25 30

Pro Lys Thr His ASN ASN Pro Tyr 45

Asn asn Glu Ile Phe Thr will Leu 60

Glu Asp Ile ASN will val val Pro 75 80

Met Pro Gly Glu Asp Leu Gly val 90 95

Asp Phe Phe Leu Lys Glu Asp Arg 105 110

-120-

<td colspan="3">Lys Ile Met</td><td colspan="2" rowspan="2">CYS asn</td><td colspan="2" rowspan="2">will Gly</td><td colspan="2" rowspan="2">Ile Lys 120</td><td rowspan="2">val</td><td colspan="3" rowspan="2">ile Ala Arg 125</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td>

<td colspan="2"></td><td> 115</td>

<td>Val</td><td>Phe</td><td>Arg</td><td>Gly</td><td>asn</td><td rowspan="2">Lys</td><td>Val</td><td rowspan="2">Lys</td><td>asn</td><td>Ala</td><td>gin</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Lys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Val</td><td>ile</td><td>Leu</td><td>tyr</td><td>will</td><td>asp</td><td>pro</td><td>Ala</td><td rowspan="2">asp</td><td>tyr</td><td>Phe</td><td>Ala</td><td>pro</td><td rowspan="2">Gly</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Lys</td><td>will</td><td>tyr</td><td>pro</td><td>asp</td><td>Gly</td><td>trp</td><td>asn</td><td>Leu</td><td>pro</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Gly</td><td>val</td><td>gin</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td> 175</td><td></td>

<td>Gly</td><td>asn</td><td>ile</td><td>Leu</td><td>asn</td><td>Leu</td><td>asn</td><td rowspan="2">Gly</td><td>Ala</td><td rowspan="2">Gly</td><td rowspan="2">asp</td><td>pro</td><td>Leu</td><td>thr</td><td>pro</td><td rowspan="2">Gly</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>tyr</td><td>pro</td><td>Ala</td><td>asn</td><td>glu</td><td>tyr</td><td>Ala</td><td>tyr</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>ile</td><td>Ala</td><td>glu</td><td>Ala</td><td>Val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Gly</td><td>Leu</td><td>pro</td><td>will</td><td>Ile</td><td>pro</td><td>val</td><td>His</td><td>pro</td><td>Ile</td><td rowspan="2">Gly</td><td>tyr</td><td rowspan="2">tyr</td><td rowspan="2">asp</td><td>Ala</td><td>gin</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>Leu</td><td>glu</td><td rowspan="2">Lys</td><td>Met</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>will</td><td>for a</td><td>pro</td><td>pro</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>trp</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>Arg</td><td>Gly</td><td>will</td><td>Leu</td><td>Lys</td><td>Val</td><td>pro</td><td rowspan="2">tyr</td><td>asn</td><td>val</td><td rowspan="2">Gly</td><td>pro</td><td rowspan="2">Gly</td><td>Phe</td><td>thr</td><td rowspan="2">Gly</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td> 255</td>

<td>asn</td><td>Phe</td><td>will</td><td>thr</td><td>n gl</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Met</td><td>It's</td><td>ile</td><td>His</td><td>will</td><td>thr</td><td>asn</td><td>glu</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Val</td><td>thr</td><td>Arg</td><td>Ile</td><td rowspan="2">tyr</td><td>asn</td><td>Val</td><td>ile</td><td rowspan="2">Gly</td><td>thr</td><td>Leu</td><td rowspan="2">Arg</td><td>Gly</td><td>Ala</td><td>val</td><td>glu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>asp</td><td rowspan="2">Arg</td><td rowspan="2">tyr</td><td>Val</td><td>ile</td><td>Leu</td><td rowspan="2">Gly</td><td>Gly</td><td>It's</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td rowspan="2">trp</td><td>val</td><td>Phe</td>

<td></td><td> 290</td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td colspan="2">Gly Gly</td><td>Ile</td><td rowspan="2">asp</td><td>pro</td><td>n gl</td><td>will</td><td rowspan="2">Gly</td><td>Ala</td><td>Ala</td><td>val</td><td>val</td><td>His</td><td>glu</td><td>ile</td><td>Val</td>

<td> 305</td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td rowspan="2">Arg</td><td>will</td><td>Phe</td><td rowspan="2">Gly</td><td>thr</td><td>Leu</td><td rowspan="2">^Theys</td><td rowspan="2">Lys</td><td>glu</td><td>GLV</td><td rowspan="2">trp</td><td>Arg</td><td>pro</td><td>Arg</td><td>View</td><td>thr</td>

<td></td><td></td><td> 325</td><td></td><td></td><td>33o</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Ile</td><td>Leu</td><td>Phe</td><td>Ala</td><td>will</td><td rowspan="2">trp</td><td rowspan="2">asp</td><td>Ala</td><td>glu</td><td>glu</td><td>Phe</td><td rowspan="2">Gly</td><td>Leu</td><td>Leu</td><td rowspan="2">Gly</td><td>will</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td> 350</td><td></td>

<td>thr</td><td>glu</td><td>trp</td><td>Ala</td><td>glu</td><td>glu</td><td>asn</td><td>will</td><td rowspan="2">Arg</td><td>Leu</td><td>Leu</td><td>gin</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>Val</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td> 365</td><td></td>

<td>Ala</td><td>tyr</td><td>Ile</td><td>asn</td><td>Ala</td><td>asp</td><td>will</td><td>will</td><td>Ile</td><td>glu</td><td>Gly</td><td>asn</td><td>tyr</td><td>thr</td><td>Leu</td><td>Arg</td>

370 375 380

-121-

<td>val</td><td>asp</td><td>CYS</td><td>thr</td><td>pro</td><td>Leu</td><td>Met</td><td>tyr</td><td>will</td><td>Leu</td><td>Val</td><td>His</td><td>asn</td><td>Leu</td><td>thr</td><td>Lys</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>glu</td><td>Leu</td><td>Lys</td><td>will</td><td>pro</td><td>asp</td><td>glu</td><td>Gly</td><td>Phe</td><td>glu</td><td>Gly</td><td>Lys</td><td>will</td><td>Leu</td><td>tyr</td><td>glu</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>will</td><td>trp</td><td>thr</td><td>Lys</td><td>Lys</td><td>will</td><td>pro</td><td>will</td><td>pro</td><td>glu</td><td>Phe</td><td>will</td><td>Gly</td><td>Met</td><td>pro</td><td>Arg</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Ile</td><td>will</td><td>Lys</td><td>Leu</td><td>Gly</td><td>will</td><td>Gly</td><td>asn</td><td>asp</td><td>Phe</td><td>glu</td><td>val</td><td>Phe</td><td>Phe</td><td>gin</td><td>Arg</td>

435

440

445

Ala Ile Leu Gly Arg Gly will Ala Arg Tyr Lys Thr Glu Thr ASN TRP 450 455 460

<td>asn</td><td>Lys</td><td>Phe</td><td>will</td><td>Gly</td><td>tyr</td><td>pro</td><td>Leu</td><td>tyr</td><td>It's</td><td>will</td><td>val</td><td>tyr</td><td>glu</td><td>thr</td><td>tyr</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>glu</td><td>Leu</td><td>val</td><td>glu</td><td>Lys</td><td>Phe</td><td>tyr</td><td>asp</td><td>pro</td><td>Met</td><td>Phe</td><td>Lys</td><td>tyr</td><td>It's</td><td>Leu</td><td>thr</td>

485

490

495

<td>val</td><td>Ala</td><td>gin</td><td>val 500</td><td>Arg</td><td>Gly</td><td>Gly</td><td>Met</td><td>Val 505</td><td>Phe</td><td>glu</td><td>Leu</td><td>Ala</td><td>asn 510</td><td>will</td><td>ile</td>

<td>val</td><td>Leu</td><td>pro</td><td>phe</td><td>asp</td><td>CYS</td><td>Arg</td><td>asp</td><td>tyr</td><td>Ala</td><td>val</td><td>val</td><td>Leu</td><td>Arg</td><td>Lys</td><td>tyr</td>

515

520

525

<td>Ala</td><td>asp</td><td>Lys</td><td>ile</td><td>tyr</td><td>will</td><td>Ile</td><td>will</td><td>Met</td><td>Lys</td><td>It's</td><td>pro</td><td>gin</td><td>glu</td><td>Met</td><td>Lys</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>thr</td><td>tyr</td><td>will</td><td>val</td><td>will</td><td>Phe</td><td>asp</td><td>will</td><td>Leu</td><td>Phe</td><td>will</td><td>Ala</td><td>val</td><td>Lys</td><td>asn</td><td>Phe</td>

545

550

555

560

Ala Glu Ile Lys Thr Phe will be Glu Asp Phe Arg Gin Leu Lys Asp 565 570 575

<td>will</td><td>asn</td><td>pro</td><td>Ile 580</td><td>val</td><td>Leu</td><td>Arg</td><td>Met</td><td>Met 585</td><td>asn</td><td>asp</td><td>gin</td><td>Leu</td><td>Met 590</td><td>Phe</td><td>Leu</td>

<td>glu</td><td>Arg</td><td>Ala</td><td>Phe</td><td>Ile</td><td>asp</td><td>pro</td><td>Leu</td><td>Gly</td><td>Leu</td><td>pro</td><td>asp</td><td>Arg</td><td>pro</td><td>Phe</td><td>tyr</td>

595

605

600

<td>Arg</td><td>His</td><td>val</td><td>Ile</td><td>tyr</td><td>Ala</td><td>pro</td><td>will</td><td>will</td><td>It's</td><td>asn</td><td>Lys</td><td>tyr</td><td>Ala</td><td>Gly</td><td>glu</td>

<td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td>

<td>will</td><td>Phe</td><td>pro</td><td>Gly</td><td>ile</td><td>tyr</td><td>asp</td><td>Ala</td><td>Leu</td><td>Phe</td><td>asp</td><td>ile</td><td>glu</td><td>will</td><td>Lys</td><td>val</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td>

<td>asp</td><td>pro</td><td>will</td><td>Lys</td><td>Ala</td><td>trp</td><td>Gly</td><td>glu</td><td>val</td><td>Lys</td><td>Arg</td><td>gin</td><td>ile</td><td>tyr</td><td>val</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

Ala Phe Thr Ala Val Ala Gin Leu Ala Glu Glu Thr will Ala val 660 665 670

-122 &lt;210&gt; 46 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 46 ggggtctaga cctcctttga ttagtatatt c 31 &lt;210&gt; 47 &lt;211&gt; 45 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 47 atcttcgcta tctgtcgccg cggcgcgtgc ttcagtttgt tgcgc &lt;210&gt; 48 &lt;211&gt; 45 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 48

-123gcgcaacaaa ctgaagcagc ggccgcggcg acagatagcg aagat &lt;210&gt; 49 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 49 tgtaggtgta tctccatgct cgagagctag gcgatcaatt tc 42 &lt;210&gt; 50 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 50 ggaattgatc gcctagctct cgagcatgga gatacaccta ca 42 &lt;210&gt; 51 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane

-124 &lt;400&gt; 51 aaacggattt atttagatcc cgggttatgg tttctgagaa ca &lt;210&gt; 52 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 52 tgttctcaga aaccataacc cgggatctaa ataaatccgt tt &lt;210&gt; 53 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 53 gggggtcgac cagctcttct tggtgaag 28 &lt;210&gt; 54 &lt;211&gt; 680 &lt;212&gt; PRT &lt;213&gt; Sztuczne

-125&lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane

<td colspan="3"> &lt;400&gt; 54</td><td colspan="3" rowspan="2">ile Met Leu 5</td><td rowspan="2">val</td><td rowspan="2">Phe</td><td rowspan="2">ile</td><td rowspan="2">thr 10</td><td rowspan="2">Leu</td><td rowspan="2">Ile</td><td rowspan="2">Leu</td><td rowspan="2">val</td><td rowspan="2">will 15</td><td rowspan="2">Leu</td>

<td>Met 1</td><td colspan="2">Lys Lys</td>

<td>pro</td><td>Ile</td><td>Ala</td><td>gin</td><td>gin</td><td>thr</td><td>glu</td><td>Ala</td><td>Lys</td><td rowspan="2">asp</td><td>Ala</td><td>will</td><td>Ala</td><td>Phe</td><td>asn</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td>

<td>glu</td><td>asn</td><td>will</td><td>ile</td><td>will</td><td>will</td><td>Met</td><td>Ala</td><td>pro</td><td>pro</td><td>Ala</td><td>will</td><td>pro</td><td>pro</td><td>Ala</td><td>will</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>pro</td><td>Lys</td><td>thr</td><td>pro</td><td>Ile</td><td>glu</td><td>Lys</td><td rowspan="2">Lys</td><td>His</td><td>Ala</td><td rowspan="2">asp</td><td>glu</td><td>Ile</td><td rowspan="2">asp</td><td rowspan="2">Lys</td><td rowspan="2">tyr</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td> 60</td><td></td>

<td>ile</td><td>gin</td><td>Gly</td><td>Leu</td><td>asp</td><td>tyr</td><td>asn</td><td rowspan="2">Lys</td><td>asn</td><td>asn</td><td>val</td><td>Leu</td><td>val</td><td rowspan="2">tyr</td><td>His</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>asp</td><td>Ala</td><td>val</td><td>thr</td><td>asn</td><td>val</td><td>pro</td><td>pro</td><td rowspan="2">Arg</td><td>Lys</td><td rowspan="2">Gly</td><td rowspan="2">tyr</td><td rowspan="2">Lys</td><td rowspan="2">asp</td><td>Gly</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td> 95</td><td></td>

<td>glu</td><td>tyr</td><td>Ile</td><td>Val</td><td>val</td><td>glu</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Lys</td><td>will</td><td>Ile</td><td>asn</td><td>gin</td><td>asn</td><td>asn</td>

-126100 105 110

<td colspan="2" rowspan="2">Ala Asp</td><td rowspan="2">Ile 115</td><td rowspan="2">gin</td><td rowspan="2">val</td><td rowspan="2">val</td><td colspan="8">Asn Ala Ile Leu Thr Tyr will be</td><td rowspan="2">pro</td><td rowspan="2">Gly</td>

<td colspan="2"> 120</td><td colspan="6"> 125</td>

<td>Ala</td><td>Leu</td><td>Val</td><td rowspan="2">Lys</td><td>Ala</td><td>asn</td><td>will</td><td>glu</td><td>Leu</td><td>val</td><td>glu</td><td>asn</td><td>gin</td><td>pro</td><td rowspan="2">asp</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>

<td>Leu</td><td>pro</td><td>Val</td><td>Lys</td><td rowspan="2">Arg</td><td>asp</td><td>will</td><td>Leu</td><td>thr</td><td>Leu</td><td>will</td><td>Ile</td><td rowspan="2">asp</td><td>Leu</td><td>pro</td><td>Gly</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Met</td><td>thr</td><td>asn</td><td>gin</td><td>asp</td><td>asn</td><td rowspan="2">Lys</td><td>Ile</td><td>val</td><td>Val</td><td rowspan="2">Lys</td><td>asn</td><td>Ala</td><td>thr</td><td>Lys</td><td>will</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asn</td><td>val</td><td>asn</td><td>asn</td><td>Ala</td><td>Val</td><td>asn</td><td>thr</td><td>Leu</td><td>val</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">trp</td><td>asn</td><td>glu</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td>tyr</td><td>Ala</td><td>gin</td><td>Ala</td><td>tyr</td><td>pro</td><td>asn</td><td>val</td><td>will</td><td>Ala</td><td>Lys</td><td>ile</td><td>asp</td><td>tyr</td><td>asp</td><td>asp</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>glu</td><td>Met</td><td>Ala</td><td rowspan="2">tyr</td><td>will</td><td>glu</td><td>will</td><td>gin</td><td>Leu</td><td>ile</td><td>Ala</td><td>Lys</td><td>Phe</td><td rowspan="2">Gly</td><td>thr</td><td>Ala</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>val</td><td>asn</td><td>asn</td><td>will</td><td>Leu</td><td>asn</td><td>val</td><td>asn</td><td>Phe</td><td rowspan="2">Gly</td><td>Ala</td><td>ile</td><td>will</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>glu</td><td>Gly</td><td>Lys</td><td>Met</td><td>gin</td><td>glu</td><td>glu</td><td>val</td><td>Ile</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>gin</td><td>Ile</td><td>tyr</td><td rowspan="2">tyr</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td>

<td>asn</td><td>Val</td><td>asn</td><td>Val</td><td>asn</td><td>glu</td><td>pro</td><td>thr</td><td>Arg</td><td>pro</td><td>will</td><td rowspan="2">Arg</td><td>Phe</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td>

<td>Ala</td><td>val</td><td>thr</td><td rowspan="2">Lys</td><td>glu</td><td>gin</td><td>Leu</td><td>gin</td><td>Ala</td><td>Leu</td><td rowspan="2">Gly</td><td>Val</td><td>asn</td><td>for a</td><td>glu</td><td>asn</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>pro</td><td>Ala</td><td rowspan="2">tyr</td><td>Ile</td><td>will</td><td>will</td><td>val</td><td>Ala</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Arg</td><td>gin</td><td>val</td><td rowspan="2">tyr</td><td>Leu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Lys</td><td>Leu</td><td>will</td><td>thr</td><td>asn</td><td>will</td><td>His</td><td>will</td><td>thr</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>Ala</td><td>Ala</td><td>Phe</td><td>asp</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td> 320</td>

<td>Ala</td><td>Ala</td><td>val</td><td>will</td><td>Gly</td><td rowspan="2">Lys</td><td>will</td><td>Val</td><td>will</td><td>Gly</td><td rowspan="2">asp</td><td>val</td><td>glu</td><td>Leu</td><td>thr</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>ile</td><td>ile</td><td rowspan="2">Lys</td><td>asn</td><td>will</td><td>will</td><td>Phe</td><td rowspan="2">Lys</td><td>Ala</td><td>val</td><td>ile</td><td rowspan="2">tyr</td><td rowspan="2">Gly</td><td>Gly</td><td>will</td><td>Ala</td>

<td></td><td></td><td> 340</td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Lys</td><td>asp</td><td>glu</td><td>val</td><td>gin</td><td>ile</td><td>Ile</td><td>asp</td><td>Gly</td><td>asn</td><td>Leu</td><td>Gly</td><td>asp</td><td>Leu</td><td rowspan="2">Arg</td><td rowspan="2">asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td>

<td>Ile</td><td>Leu</td><td>Lys</td><td>Lys</td><td>Gly</td><td>Ala</td><td>thr</td><td>Phe</td><td>asn</td><td>Arg</td><td>glu</td><td>thr</td><td>pro</td><td>Gly</td><td>Val</td><td>pro</td>

-127-

<img file="PL2155243T4_D0002.tif" />

-128645 650 ' 655

Arg Tyr Leu Glu Pro will Lys Thr Lys Arg Tyr His val val TRP Ile 660 665 670

Asp Ala Lys Thr Val ile ASN Pro 675 680 &lt;210&gt; 55 &lt;211&gt; 13 294 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 55

-129-

<td>aattccggat</td><td>gagcattcat</td><td>caggcgggca</td><td>agaatgtgaa</td><td>taaaggccgg</td><td>ataaaacttg</td><td> 60</td>

<td>tgcttatttt</td><td>tctttacggt</td><td>ctttaaaaag</td><td>gccgtaatat</td><td>ccagctgaac</td><td>ggtctggtta</td><td> 120</td>

<td>taggtacatt</td><td>gagcaactga</td><td>ctgaaatgcc</td><td>tcaaaatgtt</td><td>ctttacgatg</td><td>ccattgggat</td><td> 180</td>

<td>atatcaacgg</td><td>tggtatatcc</td><td>agtgattttt</td><td>ttctccattt</td><td>tagcttcctt</td><td>agctcctgaa</td><td> 240</td>

<td>aatctcgata</td><td>actcaaaaaa</td><td>tacgcccggt</td><td>agtgatctta</td><td>tttcattatg</td><td>gtgaaagttg</td><td> 300</td>

<td>gaacctctta</td><td>cgtgccgatc</td><td>aacgtctcat</td><td>tttcgccaaa</td><td>agttggccca</td><td>gggcttcccg</td><td> 360</td>

<td>gtatcaacag</td><td>ggacaccagg</td><td>atttatttat</td><td>tctgcgaagt</td><td>gatcttccgt</td><td>cacaggtatt</td><td> 420</td>

<td>tattcggcgc</td><td>aaagtgcgtc</td><td>gggtgatgct</td><td>gccaacttac</td><td>tgatttagtg</td><td>tatgatggtg</td><td> 480</td>

<td>tttttgaggt</td><td>gctccagtgg</td><td>cttctgtttc</td><td>tatcagctgt</td><td>ccctcctgtt</td><td>cagctactga</td><td> 540</td>

<td>cggggtggtg</td><td>cgtaacggca</td><td>aaagcaccgc</td><td>cggacatcag</td><td>cgctagcgga</td><td>gtgtatactg</td><td> 600</td>

<td>gcttactatg</td><td>ttggcactga</td><td>tgagggtgtc</td><td>agtgaagtgc</td><td>ttcatgtggc</td><td>aggagaaaaa</td><td> 660</td>

<td>aggctgcacc</td><td>ggtgcgtcag</td><td>cagaatatgt</td><td>gatacaggat</td><td>atattccgct</td><td>tcctcgctca</td><td> 720</td>

<td>ctgactcgct</td><td>acgctcggtc</td><td>gttcgactgc</td><td>ggcgagcgga</td><td>aatggcttac</td><td>gaacggggcg</td><td> 780</td>

<td>gagatttcct</td><td>ggaagatgcc</td><td>aggaagatac</td><td>ttaacaggga</td><td>agtgagaggg</td><td>ccgcggcaaa</td><td> 840</td>

<td>gccgtttttc</td><td>cataggctcc</td><td>gcccccctga</td><td>caagcatcac</td><td>gaaatctgac</td><td>gctcaaatca</td><td> 900</td>

<td>gtggtggcga</td><td>aacccgacag</td><td>gactataaag</td><td>ataccaggcg</td><td>tttccccctg</td><td>gcggctccct</td><td> 960</td>

<td>cgtgcgctct</td><td>cctgttcctg</td><td>cctttcggtt</td><td>taccggtgtc</td><td>attccgctgt</td><td>tatggccgcg</td><td> 1020</td>

<td>tttgtctcat</td><td>tccacgcctg</td><td>acactcagtt</td><td>ccgggtaggc</td><td>agttcgctcc</td><td>aagctggact</td><td> 1080</td>

<td>gtatgcacga</td><td>accccccgtt</td><td>cagtccgacc</td><td>gctgcgcctt</td><td>atccggtaac</td><td>tatcgtcttg</td><td> 1140</td>

<td>agtccaaccc</td><td>ggaaagacat</td><td>gcaaaagcac</td><td>cactggcagc</td><td>agccactggt</td><td>aattgattta</td><td> 1200</td>

<td>gaggagttag</td><td>tcttgaagtc</td><td>atgcgccggt</td><td>taaggctaaa</td><td>ctgaaaggac</td><td>aagttttggt</td><td> 1260</td>

<td>gactgcgctc</td><td>ctccaagcca</td><td>gttacctcgg</td><td>ttcaaagagt</td><td>tggtagctca</td><td>gagaaccttc</td><td> 1320</td>

<td>gaaaaaccgc</td><td>cctgcaaggc</td><td>ggttttttcg</td><td>ttttcagagc</td><td>aagagattac</td><td>gcgcagacca</td><td> 1380</td>

<td>aaacgatctc</td><td>aagaagatca</td><td>tcttattaat</td><td>cagataaaat</td><td>atttctagcc</td><td>ctcctttgat</td><td> 1440</td>

-130-

<td>tagtatattc</td><td>ctatcttaaa</td><td>gttactttta</td><td>tgtggaggca</td><td>ttaacatttg</td><td>ttaatgacgt</td><td> 1500</td>

<td>caaaaggata</td><td>gcaagactag</td><td>aataaagcta</td><td>taaagcaagc</td><td>atataatatt</td><td>gcgtttcatc</td><td> 1560</td>

<td>tttagaagcg</td><td>aatttcgcca</td><td>atattataat</td><td>tatcaaaaga</td><td>gaggggtggc</td><td>aaacggtatt</td><td> 1620</td>

<td>tggcattatt</td><td>aggttaaaaa</td><td>atgtagaagg</td><td>agagtgaaac</td><td>ccatgaaaaa</td><td>aataatgcta</td><td> 1680</td>

<td>gtttttatta</td><td>cacttatatt</td><td>agttagtcta</td><td>ccaattgcgc</td><td>aacaaactga</td><td>agcaaaggat</td><td> 1740</td>

<td>gcatctgcat</td><td>tcaataaaga</td><td>aaattcaatt</td><td>tcatccatgg</td><td>caccaccagc</td><td>atctccgcct</td><td> 1800</td>

<td>gcaagtccta</td><td>agacgccaat</td><td>cgaaaagaaa</td><td>cacgcggatg</td><td>aaatcgataa</td><td>gtatatacaa</td><td> 1860</td>

<td>ggattggatt</td><td>acaataaaaa</td><td>caatgtatta</td><td>gtataccacg</td><td>gagatgcagt</td><td>gacaaatgtg</td><td> 1920</td>

<td>ccgccaagaa</td><td>aaggttacaa</td><td>agatggaaat</td><td>gaatatattg</td><td>ttgtggagaa</td><td>aaagaagaaa</td><td> 1980</td>

<td>tccatcaatc</td><td>aaaataatgc</td><td>agacattcaa</td><td>gttgtgaatg</td><td>caatttcgag</td><td>cctaacctat</td><td> 2040</td>

<td>ccaggtgctc</td><td>tcgtaaaagc</td><td>gaattcggaa</td><td>ttagtagaaa</td><td>atcaaccaga</td><td>tgttctccct</td><td> 2100</td>

<td>gtaaaacgtg</td><td>attcattaac</td><td>actcagcatt</td><td>gatttgccag</td><td>gtatgactaa</td><td>tcaagacaat</td><td> 2160</td>

<td>aaaatagttg</td><td>taaaaaatgc</td><td>cactaaatca</td><td>aacgttaaca</td><td>acgcagtaaa</td><td>tacattagtg</td><td> 2220</td>

<td>gaaagatgga</td><td>atgaaaaata</td><td>tgctcaagct</td><td>tatccaaatg</td><td>taagtgcaaa</td><td>aattgattat</td><td> 2280</td>

<td>gatgacgaaa</td><td>tggcttacag</td><td>tgaatcacaa</td><td>ttaattgcga</td><td>aatttggtac</td><td>agcatttaaa</td><td> 2340</td>

<td>gctgtaaata</td><td>atagcttgaa</td><td>tgtaaacttc</td><td>ggcgcaatca</td><td>gtgaagggaa</td><td>aatgcaagaa</td><td> 2400</td>

<td>gaagtcatta</td><td>gttttaaaca</td><td>aatttactat</td><td>aacgtgaatg</td><td>ttaatgaacc</td><td>tacaagacct</td><td> 2460</td>

<td>tccagatttt</td><td>tcggcaaagc</td><td>tgttactaaa</td><td>gagcagttgc</td><td>aagcgcttgg</td><td>agtgaatgca</td><td> 2520</td>

<td>gaaaatcctc</td><td>ctgcatatat</td><td>ctcaagtgtg</td><td>gcgtatggcc</td><td>gtcaagttta</td><td>tttgaaatta</td><td> 2580</td>

<td>tcaactaatt</td><td>cccatagtac</td><td>taaagtaaaa</td><td>gctgcttttg</td><td>atgctgccgt</td><td>aagcggaaaa</td><td> 2640</td>

<td>tctgtctcag</td><td>gtgatgtaga</td><td>actaacaaat</td><td>atcatcaaaa</td><td>attcttcctt</td><td>caaagccgta</td><td> 2700</td>

<td>atttacggag</td><td>gttccgcaaa</td><td>agatgaagtt</td><td>caaatcatcg</td><td>acggcaacct</td><td>cggagactta</td><td> 2760</td>

<td>cgcgatattt</td><td>tgaaaaaagg</td><td>cgctactttt</td><td>aatcgagaaa</td><td>caccaggagt</td><td>tcccattgct</td><td> 2820</td>

<td>tatacaacaa</td><td>acttcctaaa</td><td>agacaatgaa</td><td>ttagctgtta</td><td>ttaaaaacaa</td><td>ctcagaatat</td><td> 2880</td>

<td>attgaaacaa</td><td>cttcaaaagc</td><td>ttatacagat</td><td>ggaaaaatta</td><td>acatcgatca</td><td>ctctggagga</td><td> 2940</td>

<td>tacgttgctc</td><td>aattcaacat</td><td>ttcttgggat</td><td>gaagtaaatt</td><td>atgatctcga</td><td>gattgtggga</td><td> 3000</td>

<td>ggctgggagt</td><td>gcgagaagca</td><td>ttcccaaccc</td><td>tggcaggtgc</td><td>ttgtggcctc</td><td>tcgtggcagg</td><td> 3060</td>

<td>gcagtctgcg</td><td>gcggtgttct</td><td>ggtgcacccc</td><td>cagtgggtcc</td><td>tcacagctgc</td><td>ccactgcatc</td><td> 3120</td>

<td>aggaacaaaa</td><td>gcgtgatctt</td><td>gctgggtcgg</td><td>cacagcctgt</td><td>ttcatcctga</td><td>agacacaggc</td><td> 3180</td>

<td>caggtatttc</td><td>aggtcagcca</td><td>cagcttccca</td><td>cacccgctct</td><td>acgatatgag</td><td>cctcctgaag</td><td> 3240</td>

<td>aatcgattcc</td><td>tcaggccagg</td><td>tgatgactcc</td><td>agccacgacc</td><td>tcatgctgct</td><td>ccgcctgtca</td><td> 3300</td>

<td>gagcctgccg</td><td>agctcacgga</td><td>tgctgtgaag</td><td>gtcatggacc</td><td>tgcccaccca</td><td>ggagccagca</td><td> 3360</td>

<td>ctggggacca</td><td>cctgctacgc</td><td>ctcaggctgg</td><td>ggcagcattg</td><td>aaccagagga</td><td>gttcttgacc</td><td> 3420</td>

<td>ccaaagaaac</td><td>ttcagtgtgt</td><td>ggacctccat</td><td>gttatttcca</td><td>atgacgtgtg</td><td>tgcgcaagtt</td><td> 3480</td>

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<td>caccctcaga</td><td>aggtgaccaa</td><td>&lt;Tt i gttcatgctg</td><td>1 11 «^ tgtgctggac</td><td colspan="2">and. · Im * i «z · gctggacagg gggcaaaagc</td><td> 3540</td>

<td>acctgctcgg</td><td>gtgattctgg</td><td>gggcccactt</td><td>gtctgttatg</td><td>gtgtgcttca</td><td>aggtatcacg</td><td> 3600</td>

<td>tcatggggca</td><td>gtgaaccatg</td><td>tgccctgccc</td><td>gaaaggcctt</td><td>ccctgtacac</td><td>caaggtggtg</td><td> 3660</td>

<td>cattaccgga</td><td>agtggatcaa</td><td>ggacaccatc</td><td>gtggccaacc</td><td>cctaaactag</td><td>tgactacaag</td><td> 3720</td>

<td>gacgatgacg</td><td>acaagtgata</td><td>cccgggatct</td><td>aaataaatcc</td><td>gtttttaaat</td><td>atgtatgcat</td><td> 3780</td>

<td>ttcttttgcg</td><td>aaatcaaaat</td><td>ttgtataata</td><td>aaatcctata</td><td>tgtaaaaaac</td><td>atcatttagc</td><td> 3840</td>

<td>gtgactttct</td><td>ttcaacagct</td><td>aacaattgtt</td><td>gttactgcct</td><td>aatgttttta</td><td>gggtatttta</td><td> 3900</td>

<td>aaaaagggcg</td><td>ataaaaaacg</td><td>attgggggat</td><td>gagacatgaa</td><td>cgctcaagca</td><td>gaagaattca</td><td> 3960</td>

<td>aaaaatattt</td><td>agaaactaac</td><td>gggataaaac</td><td>caaaacaatt</td><td>tcataaaaaa</td><td>gaacttattt</td><td> 4020</td>

<td>ttaaccaatg</td><td>ggatccacaa</td><td>gaatattgta</td><td>ttttcctata</td><td>tgatggtatc</td><td>acaaagctca</td><td> 4080</td>

<td>cgagtattag</td><td>cgagaacggg</td><td>accatcatga</td><td>atttacaata</td><td>ctacaaaggg</td><td>gctttcgtta</td><td> 4140</td>

<td>taatgtctgg</td><td>ctttattgat</td><td>acagaaacat</td><td>cggttggcta</td><td>ttataattta</td><td>gaagtcatta</td><td> 4200</td>

<td>gcgagcaggc</td><td>taccgcatac</td><td>gttatcaaaa</td><td>taaacgaact</td><td>aaaagaacta</td><td>ctgagcaaaa</td><td> 4260</td>

<td>atcttacgca</td><td>ctttttctat</td><td>gttttccaaa</td><td>ccctacaaaa</td><td>acaagtttca</td><td>tacagcctag</td><td> 4320</td>

<td>ctaaatttaa</td><td>tgatttttcg</td><td>attaacggga</td><td>agcttggctc</td><td>tatttgcggt</td><td>caacttttaa</td><td> 4380</td>

<td>tcctgaccta</td><td>tgtgtatggt</td><td>aaagaaactc</td><td>ctgatggcat</td><td>caagattaca</td><td>ctggataatt</td><td> 4440</td>

<td>taacaatgca</td><td>ggagttagga</td><td>tattcaagtg</td><td>gcatcgcaca</td><td>tagctcagct</td><td>gttagcagaa</td><td> 4500</td>

<td>ttatttccaa</td><td>attaaagcaa</td><td>gagaaagtta</td><td>tcgtgtataa</td><td>aaattcatgc</td><td>ttttatgtac</td><td> 4560</td>

<td>aaaatcgtga</td><td>ttatctcaaa</td><td>agatatgccc</td><td>ctaaattaga</td><td>tgaatggttt</td><td>tatttagcat</td><td> 4620</td>

<td>gtcctgctac</td><td>ttggggaaaa</td><td>ttaaattaaa</td><td>tcaaaaacag</td><td>tattcctcaa</td><td>tgaggaatac</td><td> 4680</td>

<td>tgttttatat</td><td>tttattcgaa</td><td>taaagaactt</td><td>acagaagcat</td><td>tttcatgaac</td><td>gcgtacgatt</td><td> 4740</td>

<td>gcttcaccaa</td><td>gaagagctgg</td><td>tcgaccgatg</td><td>cccttgagag</td><td>ccttcaaccc</td><td>agtcagctcc</td><td> 4800</td>

<td>ttccggtggg</td><td>cgcggggcat</td><td>gactatcgtc</td><td>gccgcactta</td><td>tgactgtctt</td><td>ctttatcatg</td><td> 4860</td>

<td>caactcgtag</td><td>gacaggtgcc</td><td>ggcagcgctc</td><td>tgggtcattt</td><td>tcggcgagga</td><td>ccgctttcgc</td><td> 4920</td>

<td>tggagcgcga</td><td>cgatgatcgg</td><td>cctgtcgctt</td><td>gcggtattcg</td><td>gaatcttgca</td><td>cgccctcgct</td><td> 4980</td>

<td>caagccttcg</td><td>tcactggtcc</td><td>cgccaccaaa</td><td>cgtttcggcg</td><td>agaagcaggc</td><td>cattatcgcc</td><td> 5040</td>

<td>ggcatggcgg</td><td>ccgacgcgct</td><td>gggctacgtc</td><td>ttgctggcgt</td><td>tcgcgacgcg</td><td>aggctggatg</td><td> 5100</td>

<td>gccttcccca</td><td>ttatgattct</td><td>tctcgcttcc</td><td>ggcggcatcg</td><td>ggatgcccgc</td><td>gttgcaggcc</td><td> 5160</td>

<td>atgctgtcca</td><td>ggcaggtaga</td><td>tgacgaccat</td><td>cagggacagc</td><td>ttcaaggatc</td><td>gctcgcggct</td><td> 5220</td>

<td>cttaccagcc</td><td>taacttcgat</td><td>cattggaccg</td><td>ctgatcgtca</td><td>cggcgattta</td><td>tgccgcctcg</td><td> 5280</td>

<td>gcgagcacat</td><td>ggaacgggtt</td><td>ggcatggatt</td><td>gtaggcgccg</td><td>ccctatacct</td><td>tgtctgcctc</td><td> 5340</td>

<td>cccgcgttgc</td><td>gtcgcggtgc</td><td>atggagccgg</td><td>gccacctcga</td><td>cctgaatgga</td><td>agccggcggc</td><td> 5400</td>

<td>acctcgctaa</td><td>cggattcacc</td><td>actccaagaa</td><td>ttggagccaa</td><td>tcaattcttg</td><td>cggagaactg</td><td> 5460</td>

<td>tgaatgcgca</td><td>aaccaaccct</td><td>tggcagaaca</td><td>tatccatcgc</td><td>gtccgccatc</td><td>tccagcagcc</td><td> 5520</td>

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<td>gcacgcggcg</td><td>catctcggct</td><td>ttcgatttgt</td><td>ttttgaatgg</td><td>tttatccgat</td><td>aaagaagttg</td><td> 5580</td>

<td>aagaacaaac</td><td>tggaatcaat</td><td>cgccgaacgt</td><td>ttagaaggta</td><td>tcgagcaaga</td><td>tataacgtga</td><td> 5640</td>

<td>cagtcgatca</td><td>aagaaaaaac</td><td>aatgaaaaga</td><td>gggatagtta</td><td>atgagtacgg</td><td>ttattttagc</td><td> 5700</td>

<td>tgaaaaacca</td><td>agccaggcat</td><td>tagcctacgc</td><td>aagtgcttta</td><td>aaacaaagca</td><td>ccaaaaaaga</td><td> 5760</td>

<td>cggttatttt</td><td>gagatcaaag</td><td>acccactatt</td><td>tacagatgaa</td><td>acgtttatca</td><td>cctttggttt</td><td> 5820</td>

<td>tgggcattta</td><td>gtggaattag</td><td>cagaaccagg</td><td>tcattatgac</td><td>gaaaagtggc</td><td>aaaattggaa</td><td> 5880</td>

<td>acttgaatct</td><td>ttgccgattt</td><td>ttcctgatcg</td><td>atacgatttt</td><td>gaagttgcaa</td><td>aagataaggg</td><td> 5940</td>

<td>aaagcagttt</td><td>aaaattgttg</td><td>cagaacttct</td><td>caaaaaggca</td><td>aatacaatta</td><td>ttgttgcaac</td><td> 6000</td>

<td>agatagcgac</td><td>agagaaggtg</td><td>aaaatatcgc</td><td>ctggtcgatt</td><td>atccataaag</td><td>caaatgcctt</td><td> 6060</td>

<td>ttcaaaagat</td><td>aaaacattta</td><td>aaagactatg</td><td>gatcaatagc</td><td>ttagaaaaag</td><td>atgtaatccg</td><td> 6120</td>

<td>aagcggtttt</td><td>caaaatttgc</td><td>aacctggaat</td><td>gaattactat</td><td>cccttttatc</td><td>aagaagcgca</td><td> 6180</td>

<td>aacacgccaa</td><td>attgccgatt</td><td>ggttgatcgg</td><td>catgaacgca</td><td>agccctttgt</td><td>atacgttaaa</td><td> 6240</td>

<td>tttacaacag</td><td>aagggcgtac</td><td>aaggtacatt</td><td>ttcactagga</td><td>cgtgttcaaa</td><td>cgcccacctt</td><td> 6300</td>

<td>ataccttatt</td><td>tttcagcgcc</td><td>aggaagccat</td><td>agagaatttt</td><td>aaaaaagaac</td><td>cttttttcga</td><td> 6360</td>

<td>ggtggaagct</td><td>agtataaaag</td><td>taaaccaagg</td><td>gtcgtttaag</td><td>ggcgttctaa</td><td>gccccacaca</td><td> 6420</td>

<td>gcgttttaaa</td><td>acccaagagg</td><td>agcttttagc</td><td>ttttgtttct</td><td>tctaaacaag</td><td>ctaaaatagg</td><td> 6480</td>

<td>caatcaagag</td><td>gggataattg</td><td>ctgatgttca</td><td>aaccaaagag</td><td>aagaaaacga</td><td>atagtccgag</td><td> 6540</td>

<td>tttgttttct</td><td>ttaagtagtt</td><td>tgcaatcaaa</td><td>agtcaatcag</td><td>ctttataaag</td><td>cgacagcgag</td><td> 6600</td>

<td>ccaaacttta</td><td>aaagctattt</td><td>cttttttaat</td><td>aacttaaaaa</td><td>taaacttaat</td><td>gtaacagcaa</td><td> 6660</td>

<td>gcacagtcaa</td><td>ggtatacacc</td><td>tttgacaaaa</td><td>aatagcacat</td><td>tctctatcga</td><td>aaatttttgc</td><td> 6720</td>

<td>ttatttttta</td><td>aattattttg</td><td>ggaaattttc</td><td>ccaatccctt</td><td>tttctaactc</td><td>aaaaaatata</td><td> 6780</td>

<td>atcactcaaa</td><td>atttaaaagg</td><td>gcgcacttat</td><td>acatcatttt</td><td>aaaaaattga</td><td>tgtaacgtgc</td><td> 6840</td>

<td>taagttcaaa</td><td>acaaagggcg</td><td>cacttataca</td><td>cgattttcaa</td><td>tcttgtatat</td><td>ttctaacgaa</td><td> 6900</td>

<td>aagcgtgcgc</td><td>caaaaaaccc</td><td>ccttcgtcaa</td><td>ttttgacagg</td><td>gggctttttg</td><td>atgtaaaaat</td><td> 6960</td>

<td>ttctatcgaa</td><td>atttaaaaat</td><td>tcgcttcact</td><td>catgttataa</td><td>agacttaaaa</td><td>taaaataact</td><td> 7020</td>

<td>ctttaaaatc</td><td>ttttgctagt</td><td>tgttcttcaa</td><td>tattttttat</td><td>tcggtgcatc</td><td>ttccaagtaa</td><td> 7080</td>

<td>agtataacac</td><td>actagactta</td><td>tttactacgt</td><td>ttcataagtc</td><td>attaatgcgt</td><td>gtgctctgcg</td><td> 7140</td>

<td>aggctagttt</td><td>ttgtgcaagc</td><td>acaaaaaatg</td><td>gactgaataa</td><td>atcagtccat</td><td>aagttcaaaa</td><td> 7200</td>

<td>ccaaattcaa</td><td>aatcaaaacc</td><td>acaagcaacc</td><td>aaaaaatgtg</td><td>gttgttatac</td><td>gttcataaat</td><td> 7260</td>

<td>tttatgatca</td><td>cttacgtgta</td><td>taaaattaaa</td><td>ttcactttca</td><td>aaatctaaaa</td><td>actaaatcca</td><td> 7320</td>

<td>atcatctacc</td><td>ctatgaatta</td><td>tatcttgaaa</td><td>ttcattcata</td><td>aatagtgaag</td><td>catggtaacc</td><td> 7380</td>

<td>atcacataca</td><td>gaatgatgaa</td><td>gttgcagagc</td><td>aactggtata</td><td>taaattttat</td><td>tattctcact</td><td> 7440</td>

<td>ataaaattta</td><td>cctatcgtaa</td><td>taataggcaa</td><td>taaaaagctg</td><td>ctattgttac</td><td>caatatttaa</td><td> 7500</td>

<td>attaaatgaa</td><td>ctaaaatcaa</td><td>tccaaggaat</td><td>cattgaaatc</td><td>ggtatggtgt</td><td>tttcaggtat</td><td> 7560</td>

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<td>cggtttttta</td><td>ggaaacattt</td><td>cttctttatc</td><td>tttatattca</td><td>agcaagtcat</td><td>ttttataatt</td><td> 7620</td>

<td>attataaaaa</td><td>gaaatgaagt</td><td>ttttatcaga</td><td>ttcagtccaa</td><td>atgttagtaa</td><td>atttttcagt</td><td> 7680</td>

<td>ttgcttatta</td><td>aaaactgtat</td><td>acaaaggatt</td><td>taacttatcc</td><td>caataaccta</td><td>atttattctc</td><td> 7740</td>

<td>actattaatt</td><td>cctgttctaa</td><td>acactttatt</td><td>tttatttaca</td><td>acttccataa</td><td>ttgcataaat</td><td> 7800</td>

<td>taaagaggga</td><td>taaatttcat</td><td>atcctttctt</td><td>ttttatcata</td><td>tctttaaaca</td><td>aagtaatatc</td><td> 7860</td>

<td>aatttcttta</td><td>gtaatgctat</td><td>aagtagtttg</td><td>ctgattaaaa</td><td>tagtgttcaa</td><td>aatattcttt</td><td> 7920</td>

<td>tctatcccaa</td><td>ttttctaatt</td><td>caataatatt</td><td>aaaagtcata</td><td>tataacttcc</td><td>tcctaaattt</td><td> 7980</td>

<td>taaattttta</td><td>tatttaggag</td><td>gaataatcct</td><td>ctgatttttt</td><td>catacgttat</td><td>gtcacctcgt</td><td> 8040</td>

<td>aaatattaat</td><td>tatactgaat</td><td>tagcaatttt</td><td>tatcaaataa</td><td>aacttatttt</td><td>acttccaaaa</td><td> 8100</td>

<td>cctaaattca</td><td>cgttgccaaa</td><td>aatcaatctg</td><td>cttttgcaat</td><td>tgtttttcgt</td><td>tcgcttttaa</td><td> 8160</td>

<td>agtcgatttc</td><td>attaattccg</td><td>ttaaatcaat</td><td>tggagatatt</td><td>tctctaatca</td><td>attttttaaa</td><td> 8220</td>

<td>tttagtctta</td><td>gtattcttac</td><td>ttagctttcc</td><td>ccacatactt</td><td>tcttcatgca</td><td>acaaagtata</td><td> 8280</td>

<td>aaccatagct</td><td>tgctcattaa</td><td>ttttttctaa</td><td>agtagcccac</td><td>gcaggtttca</td><td>agatgtgtaa</td><td> 8340</td>

<td>atcattaaaa</td><td>caatcattcc</td><td>agtaatcaac</td><td>catatctctt</td><td>tttaattcaa</td><td>cttctacacg</td><td> 8400</td>

<td>ccataaatgt</td><td>tcagacacaa</td><td>cttcaacatc</td><td>tgcgttatct</td><td>ttacgttctt</td><td>gttttttatt</td><td> 8460</td>

<td>ataaattcta</td><td>ataaatctat</td><td>cactatcacg</td><td>gacaccaaaa</td><td>tattttgttt</td><td>ctggcttgcc</td><td> 8520</td>

<td>attacgacca</td><td>taaaaaacag</td><td>ttttcttaac</td><td>tgctttatca</td><td>gtcattgcat</td><td>agtaatcgct</td><td> 8580</td>

<td>caaatcatct</td><td>tcaaaatcaa</td><td>aagctaagtc</td><td>taatcttgta</td><td>aaaccgtcat</td><td>cttccatgta</td><td> 8640</td>

<td>gtcgataata</td><td>ttttgtttta</td><td>accaaatcat</td><td>ttcttcatgt</td><td>gtgagtttat</td><td>tgggattaaa</td><td> 8700</td>

<td>ttcaacacgc</td><td>atattacgtc</td><td>tatcccaagt</td><td>atctgctttt</td><td>actttgtcat</td><td>attcgatata</td><td> 8760</td>

<td>aactttttct</td><td>tgaagtgcct</td><td>tagctttaaa</td><td>ctttgtttga</td><td>agtatatccc</td><td>aaagtcgtat</td><td> 8820</td>

<td>ttgtggctct</td><td>acactcataa</td><td>agtcagatag</td><td>ctttttagca</td><td>ttagttttgt</td><td>tcaaatttcc</td><td> 8880</td>

<td>aacgattgtc</td><td>atggcatcaa</td><td>aacttaatgc</td><td>gggttgagat</td><td>tttcccaaag</td><td>tttgaccact</td><td> 8940</td>

<td>taaccggcta</td><td>ttacttaacc</td><td>ggctattaga</td><td>gacggaacta</td><td>actcaacgct</td><td>agtagtggat</td><td> 9000</td>

<td>ttaatcccaa</td><td>atgagccaac</td><td>agaaccagaa</td><td>ccagaaacag</td><td>aacaagtaac</td><td>attggagtta</td><td> 9060</td>

<td>gaaatggaag</td><td>aagaaaaaag</td><td>caatgatttc</td><td>gtgtgaataa</td><td>tgcacgaaat</td><td>cattgcttat</td><td> 9120</td>

<td>ttttttaaaa</td><td>agcgatatac</td><td>tagatataac</td><td>gaaacaacga</td><td>actgaataaa</td><td>gaatacaaaa</td><td> 9180</td>

<td>aaagagccac</td><td>gaccagttaa</td><td>agcctgagaa</td><td>actttaactg</td><td>cgagccttaa</td><td>ttgattacca</td><td> 9240</td>

<td>ccaatcaatt</td><td>aaagaagtcg</td><td>agacccaaaa</td><td>tttggtaaag</td><td>tatttaatta</td><td>ctttattaat</td><td> 9300</td>

<td>cagatactta</td><td>aatatctgta</td><td>aacccattat</td><td>atcgggtttt</td><td>tgaggggatt</td><td>tcaagtcttt</td><td> 9360</td>

<td>aagaagatac</td><td>caggcaatca</td><td>attaagaaaa</td><td>acttagttga</td><td>ttgccttttt</td><td>tgttgtgatt</td><td> 9420</td>

<td>caactttgat</td><td>cgtagcttct</td><td>aactaattaa</td><td>ttttcgtaag</td><td>aaaggagaac</td><td>agctgaatga</td><td> 9480</td>

<td>atatcccttt</td><td>tgttgtagaa</td><td>actgtgcttc</td><td>atgacggctt</td><td>gttaaagtac</td><td>aaatttaaaa</td><td> 9540</td>

<td>atagtaaaat</td><td>tcgctcaatc</td><td>actaccaagc</td><td>caggtaaaag</td><td>taaaggggct</td><td>atttttgcgt</td><td> 9600</td>

-134-

<td>atcgctcaaa</td><td>aaaaagcatg</td><td>attggcggac</td><td>gtggcgttgt</td><td>tctgacttcc</td><td>gaagaagcga</td><td> 9660</td>

<td>ttcacgaaaa</td><td>tcaagataca</td><td>tttacgcatt</td><td>ggacaccaaa</td><td>cgtttatcgt</td><td>tatggtacgt</td><td> 9720</td>

<td>atgcagacga</td><td>aaaccgttca</td><td>tacactaaag</td><td>gacattctga</td><td>aaacaattta</td><td>agacaaatca</td><td> 9780</td>

<td>ataccttctt</td><td>tattgatttt</td><td>gatattcaca</td><td>cggaaaaaga</td><td>aactatttca</td><td>gcaagcgata</td><td> 9840</td>

<td>ttttaacaac</td><td>agctattgat</td><td>ttaggtttta</td><td>tgcctacgtt</td><td>aattatcaaa</td><td>tctgataaag</td><td> 9900</td>

<td>gttatcaagc</td><td>atattttgtt</td><td>ttagaaacgc</td><td>cagtctatgt</td><td>gacttcaaaa</td><td>tcagaattta</td><td> 9960</td>

<td>aatctgtcaa</td><td>agcagccaaa</td><td>ataatctcgc</td><td>aaaatatccg</td><td>agaatatttt</td><td>ggaaagtctt</td><td> 10020</td>

<td>tgccagttga</td><td>tctaacgtgc</td><td>aatcattttg</td><td>ggattgctcg</td><td>tataccaaga</td><td>acggacaatg</td><td> 10080</td>

<td>tagaattttt</td><td>tgatcccaat</td><td>taccgttatt</td><td>ctttcaaaga</td><td>atggcaagat</td><td>tggtctttca</td><td> 10140</td>

<td>aacaaacaga</td><td>taataagggc</td><td>tttactcgtt</td><td>caagtctaac</td><td>ggttttaagc</td><td>ggtacagaag</td><td> 10200</td>

<td>gcaaaaaaca</td><td>agtagatgaa</td><td>ccctggttta</td><td>atctcttatt</td><td>gcacgaaacg</td><td>aaattttcag</td><td> 10260</td>

<td>gagaaaaggg</td><td>tttagtaggg</td><td>cgcaatagcg</td><td>ttatgtttac</td><td>cctctcttta</td><td>gcctacttta</td><td> 10320</td>

<td>gttcaggcta</td><td>ttcaatcgaa</td><td>acgtgcgaat</td><td>ataatatgtt</td><td>tgagtttaat</td><td>aatcgattag</td><td> 10380</td>

<td>atcaaccctt</td><td>agaagaaaaa</td><td>gaagtaatca</td><td>aaattgttag</td><td>aagtgcctat</td><td>tcagaaaact</td><td> 10440</td>

<td>atcaaggggc</td><td>taatagggaa</td><td>tacattacca</td><td>ttctttgcaa</td><td>agcttgggta</td><td>tcaagtgatt</td><td> 10500</td>

<td>taaccagtaa</td><td>agatttattt</td><td>gtccgtcaag</td><td>ggtggtttaa</td><td>attcaagaaa</td><td>aaaagaagcg</td><td> 10560</td>

<td>aacgtcaacg</td><td>tgttcatttg</td><td>tcagaatgga</td><td>aagaagattt</td><td>aatggcttat</td><td>attagcgaaa</td><td> 10620</td>

<td>aaagcgatgt</td><td>atacaagcct</td><td>tatttagcga</td><td>cgaccaaaaa</td><td>agagattaga</td><td>gaagtgctag</td><td> 10680</td>

<td>gcattcctga</td><td>acggacatta</td><td>gataaattgc</td><td>tgaaggtact</td><td>gaaggcgaat</td><td>caggaaattt</td><td> 10740</td>

<td>tctttaagat</td><td>taaaccagga</td><td>agaaatggtg</td><td>gcattcaact</td><td>tgctagtgtt</td><td>aaatcattgt</td><td> 10800</td>

<td>tgctatcgat</td><td>cattaaatta</td><td>aaaaaagaag</td><td>aacgagaaag</td><td>ctatataaag</td><td>gcgctgacag</td><td> 10860</td>

<td>cttcgtttaa</td><td>tttagaacgt</td><td>acatttattc</td><td>aagaaactct</td><td>aaacaaattg</td><td>gcagaacgcc</td><td> 10920</td>

<td>ccaaaacgga</td><td>cccacaactc</td><td>gatttgttta</td><td>nrtaroatar J -------</td><td>annrtoaaaa - --------</td><td>taaaacccgc</td><td> 10980</td>

<td>actatgccat</td><td>tacatttata</td><td>tctatgatac</td><td>gtgtttgttt</td><td>ttctttgctg</td><td>tttagtgaat</td><td> 11040</td>

<td>gattagcaga</td><td>aatatacaga</td><td>gtaagatttt</td><td>aattaattat</td><td>tagggggaga</td><td>aggagagagt</td><td> 11100</td>

<td>agcccgaaaa</td><td>cttttagttg</td><td>gcttggactg</td><td>aacgaagtga</td><td>gggaaaggct</td><td>actaaaacgt</td><td> 11160</td>

<td>cgaggggcag</td><td>tgagagcgaa</td><td>gcgaacactt</td><td>gatcttttaa</td><td>gttgctatca</td><td>tttataggtc</td><td> 11220</td>

<td>aatagagtat</td><td>acctatttgt</td><td>cctaatatga</td><td>ttttagcagt</td><td>ataattgact</td><td>tggtgaatag</td><td> 11280</td>

<td>gtcatttaag</td><td>ttgggcataa</td><td>taggaggagt</td><td>aaaatgaaaa</td><td>aatttattta</td><td>tcgagtttta</td><td> 11340</td>

<td>gaaaatgacg</td><td>aagtggtggc</td><td>tatttttaat</td><td>gagcaacaat</td><td>atgcgcaaga</td><td>ttttatcgct</td><td> 11400</td>

<td>tacgaaaaga</td><td>caatttctga</td><td>taagcaattt</td><td>gaaattgaaa</td><td>aagtagatat</td><td>tgctgattgg</td><td> 11460</td>

<td>ttattgcaac</td><td>cgagagaatt</td><td>ttagaggttg</td><td>gttgaaaatg</td><td>gctaaaattg</td><td>gttatgcacg</td><td> 11520</td>

<td>tgtcagtagc</td><td>aaagaacaga</td><td>acttagatcg</td><td>gcaattacaa</td><td>gcgttacagg</td><td>gcgtttctaa</td><td> 11580</td>

<td>ggtcttttca</td><td>gacaaattaa</td><td>gcggtcaatc</td><td>ggtcgaacgc</td><td>ccacaattac</td><td>aagctatgct</td><td> 11640</td>

-135-

<td>taactatatt</td><td>cgtgaagggg</td><td>atattgttat</td><td>tgttaćtgaa</td><td>ttagatcgat</td><td>taggacgaaa</td><td> 11700</td>

<td>taataaagaa</td><td>ttaacagaat</td><td>tgatgaatca</td><td>aattcaaatt</td><td>aagggggcaa</td><td>ccctggaagt</td><td> 11760</td>

<td>cttaaattta</td><td>ccctcaatga</td><td>atggtattga</td><td>agatgaaaat</td><td>ttaaggcgtt</td><td>tgattaatag</td><td> 11820</td>

<td>ccttgtcatt</td><td>gaattgtaca</td><td>agtatcaagc</td><td>agaatcagaa</td><td>cgaaaaaaaa</td><td>ttaaggaacg</td><td> 11880</td>

<td>tcaggcacaa</td><td>ggaatcgaaa</td><td>ttgctaagaa</td><td>aaaaggcaaa</td><td>ttcaaaggtc</td><td>gtcagcataa</td><td> 11940</td>

<td>atttaaagaa</td><td>aatgatccac</td><td>gtttaaagtc</td><td>gggcagcgtt</td><td>gggtcctggc</td><td>cacgggtgcg</td><td> 12000</td>

<td>catgatcgtg</td><td>ctcctgtcgt</td><td>tgaggacccg</td><td>gctaggctgg</td><td>cggggttgcc</td><td>ttactggtta</td><td> 12060</td>

<td>gcagaatgaa</td><td>tcaccgatac</td><td>gcgagcgaac</td><td>gtgaagcgac</td><td>tgctgctgca</td><td>aaacgtctgc</td><td> 12120</td>

<td>gacctgagca</td><td>acaacatgaa</td><td>tggtcttcgg</td><td>tttccgtgtt</td><td>tcgtaaagtc</td><td>tggaaacgcg</td><td> 12180</td>

<td>gaagtcccct</td><td>acgtgctgct</td><td>gaagttgccc</td><td>gcaacagaga</td><td>gtggaaccaa</td><td>ccggtgatac</td><td> 12240</td>

<td>cacgatacta</td><td>tgactgagag</td><td>tcaacgccat</td><td>gagcggcctc</td><td>atttcttatt</td><td>ctgagttaca</td><td> 12300</td>

<td>acagtccgca</td><td>ccgctgccgg</td><td>tagctccttc</td><td>cggtgggcgc</td><td>ggggcatgac</td><td>tatcgtcgcc</td><td> 12360</td>

<td>gcacttatga</td><td>ctgtcttctt</td><td>tatcatgcaa</td><td>ctcgtaggac</td><td>aggtgccggc</td><td>agcgcccaac</td><td> 12420</td>

<td>agtcccccgg</td><td>ccacggggcc</td><td>tgccaccata</td><td>cccacgccga</td><td>aacaagcgcc</td><td>ctgcaccatt</td><td> 12480</td>

<td>atgttccgga</td><td>tctgcatcgc</td><td>aggatgctgc</td><td>tggctaccct</td><td>gtggaacacc</td><td>tacatctgta</td><td> 12540</td>

<td>ttaacgaagc</td><td>gctaaccgtt</td><td>tttatcaggc</td><td>tctgggaggc</td><td>agaataaatg</td><td>atcatatcgt</td><td> 12600</td>

<td>caattattac</td><td>ctccacgggg</td><td>agagcctgag</td><td>caaactggcc</td><td>tcaggcattt</td><td>gagaagcaca</td><td> 12660</td>

<td>cggtcacact</td><td>gcttccggta</td><td>gtcaataaac</td><td>cggtaaacca</td><td>gcaatagaca</td><td>taagcggcta</td><td> 12720</td>

<td>tttaacgacc</td><td>ctgccctgaa</td><td>ccgacgaccg</td><td>ggtcgaattt</td><td>gctttcgaat</td><td>ttctgccatt</td><td> 12780</td>

<td>catccgctta</td><td>ttatcactta</td><td>ttcaggcgta</td><td>gcaaccaggc</td><td>gtttaagggc</td><td>accaataact</td><td> 12840</td>

<td>gccttaaaaa</td><td>aattacgccc</td><td>cgccctgcca</td><td>ctcatcgcag</td><td>tactgttgta</td><td>attcattaag</td><td> 12900</td>

<td>cattctgccg</td><td>acatggaagc</td><td>catcacaaac</td><td>ggcatgatga</td><td>acctgaatcg</td><td>ccagcggcat</td><td> 12960</td>

<td>cagcaccttg</td><td>tcgccttgcg</td><td>tataatattt</td><td>gcccatggtg</td><td>aaaacggggg</td><td>cgaagaagtt</td><td> 13020</td>

<td>gtccatattg</td><td>gccacgttta</td><td>aatcaaaact</td><td>ggtgaaactc</td><td>acccagggat</td><td>tggctgagac</td><td> 13080</td>

<td>gaaaaacata</td><td>ttctcaataa</td><td>accctttagg</td><td>gaaataggcc</td><td>aggttttcac</td><td>cgtaacacgc</td><td> 13140</td>

<td>cacatcttgc</td><td>gaatatatgt</td><td>gtagaaactg</td><td>ccggaaatcg</td><td>tcgtggtatt</td><td>cactccagag</td><td> 13200</td>

<td>cgatgaaaac</td><td>gtttcagttt</td><td>gctcatggaa</td><td>aacggtgtaa</td><td>caagggtgaa</td><td>cactatccca</td><td> 13260</td>

<td>tatcaccagc</td><td>tcaccgtctt</td><td>tcattgccat</td><td>acgg</td><td></td><td></td><td> 13294</td>

&lt;210&gt; 56 &lt;211&gt; 368 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes

-136&lt;400&gt; 56

Val Thr Met His Arg Gly trp TRP Pro Ile Glu Ile Thr Arg Ala Asp 15 10 15

-137-

<td>Ala</td><td>ile</td><td>Arg</td><td>glu 20</td><td>asn</td><td>ile</td><td>Lys</td><td>asn</td>

<td>val</td><td>asp</td><td>Leu 35</td><td>trp</td><td>Ala</td><td>val</td><td>val</td><td>Lys 40</td>

<td>ile</td><td>glu 50</td><td>val</td><td>Ala</td><td>Arg</td><td>thr</td><td>Ala 55</td><td>Lys</td>

<td>val 65</td><td>Ala</td><td>ile</td><td>Leu</td><td>asp</td><td>glu 70</td><td>Ala</td><td>Leu</td>

<td>asp</td><td>asp</td><td>Phe</td><td>Ile</td><td>Leu 85</td><td>Val</td><td>Leu</td><td>Gly</td>

<td>Leu</td><td>Ala</td><td>Ala</td><td>Lys 100</td><td>asn</td><td>His</td><td>Ile</td><td>will</td>

<td>Leu</td><td>glu</td><td>asn 115</td><td>Leu</td><td>thr</td><td>Leu</td><td>glu</td><td>Ala 120</td>

<td>asp</td><td>will 130</td><td>Gly</td><td>Met</td><td>Gly</td><td>Arg</td><td>Leu 135</td><td>Gly</td>

<td>Arg 145</td><td>Ile</td><td>glu</td><td>Ala</td><td>thr</td><td>will 150</td><td>thr</td><td>asn</td>

<td>Ile</td><td>tyr</td><td>thr</td><td>His</td><td>Phe 165</td><td>Ala</td><td>thr</td><td>Ala</td>

<td>glu</td><td>gin</td><td>gin</td><td>Leu 180</td><td>Ala</td><td>Lys</td><td>Phe</td><td>gin</td>

<td>Arg</td><td>pro</td><td>thr 195</td><td>tyr</td><td>val</td><td>His</td><td>thr</td><td>Ala 200</td>

<td>pro</td><td>gin 210</td><td>Ile</td><td>Gly</td><td>Phe</td><td>asp</td><td>Ala 215</td><td>Ile</td>

<td>Leu 225</td><td>thr</td><td>pro</td><td>will</td><td>thr</td><td>glu 230</td><td>Ile</td><td>Lys</td>

<td>pro</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Leu 245</td><td>tyr</td><td>thr</td><td>glu</td>

<td>pro</td><td>Gly</td><td>asp</td><td>will 260</td><td>val</td><td>will</td><td>tyr</td><td>Gly</td>

<td>glu</td><td>trp</td><td>val</td><td>Ala</td><td>thr</td><td>Leu</td><td>pro</td><td>Ile</td>

275 280

<td>glu 25</td><td>gin</td><td>asn</td><td>Lys</td><td>Leu</td><td>pro 30</td><td>glu</td><td>will</td>

<td>Ala</td><td>asn</td><td>Ala</td><td>tyr</td><td>Gly 45</td><td>His</td><td>Gly</td><td>Ile</td>

<td>glu</td><td>Ala</td><td>Gly</td><td>Ala 60</td><td>Lys</td><td>Gly</td><td>Phe</td><td>CYS</td>

<td>Ala</td><td>Leu</td><td>Arg 75</td><td>glu</td><td>Ala</td><td>Gly</td><td>Phe</td><td>gin 80</td>

<td>Ala</td><td>thr 90</td><td>Arg</td><td>Lys</td><td>glu</td><td>asp</td><td>Ala 95</td><td>asn</td>

<td>Leu 105</td><td>thr</td><td>val</td><td>Phe</td><td>Arg</td><td>glu 110</td><td>ASP</td><td>trp</td>

<td>thr</td><td>Leu</td><td>Arg</td><td>ile</td><td>His 125</td><td>Leu</td><td>Lys</td><td>val</td>

<td>ile</td><td>Arg</td><td>thr</td><td>thr 140</td><td>glu</td><td>glu</td><td>Ala</td><td>Arg</td>

<td>asp</td><td>His</td><td>Gl 155 n</td><td>Leu</td><td>gin</td><td>Leu</td><td>glu</td><td>Gly 160</td>

<td>asp</td><td>gin 170</td><td>Leu</td><td>glu</td><td>thr</td><td>will</td><td>tyr 175</td><td>Phe</td>

<td>thr 185</td><td>Ile</td><td>Leu</td><td>thr</td><td>will</td><td>Leu 190</td><td>Lys</td><td>Lys</td>

<td>asn</td><td>will</td><td>Ala</td><td>Ala</td><td>will 205</td><td>Leu</td><td>Leu</td><td>gin</td>

<td>Arg</td><td>Phe</td><td>Gly</td><td>ile 220</td><td>will</td><td>Met</td><td>tyr</td><td>Gly</td>

<td>thr</td><td>will</td><td>Leu 235</td><td>pro</td><td>Phe</td><td>glu</td><td>Leu</td><td>Lys 240</td>

<td>Met</td><td>val 250</td><td>His</td><td>val</td><td>Lys</td><td>glu</td><td>Leu 255</td><td>Ala</td>

<td>Ala 265</td><td>thr</td><td>tyr</td><td>thr</td><td>Ala</td><td>thr 270</td><td>glu</td><td>Arg</td>

<td>Gly</td><td>tyr</td><td>Ala</td><td>asp</td><td>Gly</td><td>Leu</td><td>ile</td><td>Arg</td>

285

-138-

<td>His</td><td colspan="2">Tyr will 290</td><td>Gly</td><td>Phe</td><td>It's</td><td>val 295</td><td>Leu</td><td>Val</td><td>asp</td><td colspan="3">Gly Glu Pro 300</td><td>Ala</td><td>pro</td><td>Ile</td>

<td>Ile</td><td>Gly</td><td rowspan="2">Arg</td><td>Val</td><td rowspan="2">CYS</td><td>Met</td><td rowspan="2">ASP</td><td>gin</td><td>thr</td><td>Ile</td><td>ile</td><td>Lys</td><td>Leu</td><td>pro</td><td>Arg</td><td>glu</td>

<td> 305</td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Phe</td><td>gin</td><td>thr</td><td rowspan="2">Gly</td><td>will</td><td rowspan="2">Lys</td><td>val</td><td>thr</td><td>ile</td><td>Ile</td><td rowspan="2">Gly</td><td>Lys</td><td>asp</td><td>His</td><td>Gly</td><td>asn</td>

<td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>thr</td><td>val</td><td>thr</td><td>Ala</td><td rowspan="2">asp</td><td rowspan="2">asp</td><td>Ala</td><td>Ala</td><td>n gl</td><td rowspan="2">tyr</td><td>Leu</td><td>asp</td><td>thr</td><td>ile</td><td>asn</td><td>tyr</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>glu</td><td>val</td><td>thr</td><td rowspan="2">CYS</td><td>Leu</td><td>Leu</td><td>asn</td><td>glu</td><td rowspan="2">Arg</td><td>Ile</td><td>pro</td><td rowspan="2">Arg</td><td>Lys</td><td>tyr</td><td>ile</td><td>His</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

&lt;210&gt; 57 &lt;211&gt; 289 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 57

-139Met Lys 1

Val Asp

Val val

Asp Arg 50

Will Lys 65

ile asn

Pro asn

Leu thr

Glu Gly 130

Ile Asp 145

His ile val Ala Leu Glu 20

Leu Arg 35

Tyr Leu

glu glu

asn thr

ASN Arg 100

Ala Ala 115

thr Gly

Lys will

gin gin

Val ASN 5

asp Arg

tyr ASN

Ala will

Leu Arg 70

ASN 85 Gly

His val

Ala Arg

Ala Ile

ASN Leu 150

Ala asn

His asn

Gly Tyr

Gly Lys 40

Ala Ala 55

Leu glu

Val Tyr Pro ile

Glu 120 val

Glu Thr 135

Leu Leu

Leu Glu

Leu val 10

Gin Phe 25

Phe Phe

Lys ile

Leu Glu

Leu 90 GLN

Asp Asp 105

pro Arg

glu Asp

Gly ASN

Ala Ile

glu Arg

Gly Asp

thr Tyr

Leu Asp 60

Leu Lys Thr 75 val

Phe Pro

Val asn Glu Leu Arg 140 ile 155

His Leu

glu Asp

Gly val 30

Asn Glu Ile 45 val

Val Ala

Arg Gly

Leu Glu 110

Gin Arg 125

trp Leu

Ala Lys

Arg Gly

Thr 15 Ala

tyr Glu

His ile

pro Tyr

Glu Gin val ASN 80 95

Gly val

Phe val

Arg CYS

Lys 160 asn

glu Gin

-140-

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td>r *</td><td></td><td> 170</td><td>I "»</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>thr</td><td>glu</td><td>CYS</td><td>will</td><td>Ala</td><td>will</td><td>asn</td><td>val</td><td>will</td><td>ile</td><td>ile</td><td rowspan="2">Lys</td><td>ASP</td><td rowspan="2">Gly</td><td>val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td> 190</td><td></td>

<td>Leu</td><td rowspan="2">trp</td><td>thr</td><td>His</td><td>Ala</td><td>Ala</td><td rowspan="2">asp</td><td>asn</td><td>Leu</td><td>ile</td><td>Leu</td><td>asn</td><td>Gly</td><td>Ile</td><td>thr</td><td>Arg</td>

<td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>gin</td><td>val</td><td>Ile</td><td>ile</td><td rowspan="2">asp</td><td>val</td><td>Ala</td><td rowspan="2">Lys</td><td rowspan="2">Lys</td><td>asn</td><td>Gly</td><td>ile</td><td>pro</td><td>val</td><td>Lys</td><td>glu</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td rowspan="2">asp</td><td>Phe</td><td>thr</td><td>Leu</td><td>thr</td><td rowspan="2">asp</td><td>Leu</td><td rowspan="2">Arg</td><td>glu</td><td>Ala</td><td rowspan="2">asp</td><td>glu</td><td>val</td><td>Phe</td><td>ile</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>will</td><td>will</td><td>thr</td><td>thr</td><td>ile</td><td>glu</td><td>Ile</td><td>thr</td><td>pro</td><td>Ile</td><td>thr</td><td>His</td><td>Ile</td><td rowspan="2">asp</td><td>Gly</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td><td></td>

<td>gin</td><td>val</td><td>Ala</td><td>asp</td><td rowspan="2">Gly</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>pro</td><td>ile</td><td>thr</td><td>Ala</td><td>gin</td><td>Leu</td><td>His</td><td>gin</td>

<td></td><td></td><td></td><td> 260</td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td rowspan="2">tyr</td><td>Phe</td><td>val</td><td>glu</td><td>glu</td><td>Ile</td><td>thr</td><td>Arg</td><td>Ala</td><td rowspan="2">CYS</td><td rowspan="2">Gly</td><td>glu</td><td>Leu</td><td>glu</td><td>Phe</td><td>Ala</td>

<td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

Lys &lt;210&gt; 58 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Sztuczne &lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 58 gtgctcgaga ttgtgggagg ctgggagtg 29 &lt;210&gt; 59 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Sztuczne

-141&lt;220&gt;

&lt;223&gt; chemicznie syntetyzowane &lt;400&gt; 59 gatactagtt taggggttgg ccacgatgg 29 &lt;210&gt; 60 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 60

His CYS ile ile val will Lys Arg Leu ASN 15 10 &lt;210&gt; 61 &lt;211&gt; 390 &lt;212&gt; PRT &lt;213&gt; Listeria monocytogenes &lt;400&gt; 61

-142-

<td>Met 1</td><td colspan="2">Ala Arg</td><td colspan="2">Met Met 5</td><td>Val</td><td>Val</td><td colspan="2">phe ile</td><td colspan="4">Thr 10 Ala ASN CYS</td><td>Ile</td><td>thr 15</td><td>ile</td>

<td>asn</td><td>pro</td><td rowspan="2">asp</td><td>Ile</td><td>ile</td><td>Phe</td><td>Ala</td><td>Ala</td><td>thr</td><td rowspan="2">ASP</td><td>will</td><td>glu</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Leu</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>asn</td><td>thr</td><td>asp</td><td>glu</td><td rowspan="2">trp</td><td>glu</td><td>glu</td><td>glu</td><td rowspan="2">Lys</td><td>thr</td><td>glu</td><td>glu</td><td>gin</td><td>pro</td><td>will</td><td>glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>asn</td><td>thr</td><td>Gly</td><td>pro</td><td rowspan="2">Arg</td><td>tyr</td><td>glu</td><td>thr</td><td>Ala</td><td rowspan="2">Arg</td><td>glu</td><td>val</td><td>will</td><td>will</td><td rowspan="2">Arg</td>

<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>asp</td><td>Ile</td><td>glu</td><td>glu</td><td>Leu</td><td>glu</td><td rowspan="2">Lys</td><td>will</td><td>asn</td><td rowspan="2">Lys</td><td>val</td><td rowspan="2">Lys</td><td>asn</td><td>thr</td><td>asn</td><td>Lys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>Ala</td><td>asp</td><td>Leu</td><td>Ile</td><td>Ala</td><td>Met</td><td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>Lys</td><td>Ala</td><td>glu</td><td>Lys</td><td>Gly</td><td>pro</td><td>asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>asn</td><td>asn</td><td>asn</td><td>asn</td><td>asn</td><td rowspan="2">Gly</td><td>glu</td><td>gin</td><td>thr</td><td rowspan="2">Gly</td><td>asn</td><td>val</td><td>Ala</td><td>ile</td><td>asn</td><td>glu</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>glu</td><td>Ala</td><td>will</td><td rowspan="2">Gly</td><td>val</td><td rowspan="2">asp</td><td rowspan="2">Arg</td><td>pro</td><td>thr</td><td>Leu</td><td>gin</td><td>val</td><td>glu</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>His</td>

<td></td><td></td><td> 115</td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td>

<td>pro</td><td>Gly</td><td>Leu</td><td>will</td><td>will</td><td>asp</td><td>will</td><td>Ala</td><td>Ala</td><td>glu</td><td>Ile</td><td>Lys</td><td>Lys</td><td rowspan="2">Arg</td><td>Arg</td><td>Lys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>

<td>Ala</td><td>ile</td><td>Ala</td><td>will</td><td>will</td><td>asp</td><td>will</td><td>glu</td><td>Leu</td><td>glu</td><td>will</td><td>Leu</td><td>thr</td><td rowspan="2">tyr</td><td>pro</td><td>asp</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>

<td>Lys</td><td>pro</td><td>thr</td><td>Lys</td><td>Ala</td><td>asn</td><td rowspan="2">Lys</td><td rowspan="2">Arg</td><td>Lys</td><td>val</td><td>for a</td><td rowspan="2">Lys</td><td>glu</td><td>will</td><td>val</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td> 175</td><td></td>

<td>asp</td><td>Ala</td><td>will</td><td>glu</td><td>will</td><td rowspan="2">asp</td><td>Leu</td><td rowspan="2">asp</td><td>will</td><td>will</td><td>Met</td><td>gin</td><td>will</td><td>Ala</td><td rowspan="2">asp</td><td>glu</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td>

<td>will</td><td>thr</td><td>pro</td><td>gin</td><td>pro</td><td>Leu</td><td rowspan="2">Lys</td><td>Ala</td><td>asn</td><td>gin</td><td rowspan="2">Lys</td><td>pro</td><td>Phe</td><td>Phe</td><td>pro</td><td rowspan="2">Lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>val</td><td>Phe</td><td>Lys</td><td>Lys</td><td>Ile</td><td>Lys</td><td>asp</td><td>Ala</td><td>Gly</td><td>Lys</td><td>trp</td><td>val</td><td>Arg</td><td>asp</td><td>Lys</td><td>ile</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>asp</td><td>glu</td><td>asn</td><td>pro</td><td>glu</td><td>val</td><td>Lys</td><td>Lys</td><td>Ala</td><td>ile</td><td>val</td><td rowspan="2">ASP</td><td rowspan="2">Lys</td><td>will</td><td>Ala</td><td>Gly</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td> 240</td>

-143-

<td colspan="4">Ile Asp Gin Leu</td><td>Leu 245</td><td colspan="3">Leu Lys Thr</td><td>Lys</td><td>Lys 250</td><td colspan="4">Will Glu Glu Val</td><td>asn 255</td><td>Ala</td>

<td>will</td><td rowspan="2">asp</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>asp</td><td>glu</td><td>glu</td><td>Leu</td><td rowspan="2">Arg</td><td>Leu</td><td>Ala</td><td>Leu</td>

<td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>pro</td><td>glu</td><td>thr</td><td>pro</td><td>Met</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Phe</td><td>asn</td><td>Ala</td><td>pro</td><td>thr</td><td>pro</td><td>will</td><td>glu</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>pro</td><td>will</td><td>will</td><td>Phe</td><td>glu</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>asp</td><td>glu</td><td>glu</td><td>Leu</td><td rowspan="2">Arg</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Leu</td><td>pro</td><td>glu</td><td>thr</td><td>pro</td><td>Met</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Phe</td><td>asn</td><td>Ala</td><td>pro</td><td>Ala</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>thr</td><td>will</td><td>glu</td><td>pro</td><td>will</td><td>will</td><td>Phe</td><td>glu</td><td>Phe</td><td>pro</td><td>pro</td><td>pro</td><td>pro</td><td>thr</td><td>glu</td><td rowspan="2">asp</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td>

<td>glu</td><td>Leu</td><td>glu</td><td>ile</td><td>Met</td><td rowspan="2">Arg</td><td>glu</td><td>thr</td><td>Ala</td><td>pro</td><td>will</td><td>Leu</td><td>asp</td><td>will</td><td>will</td><td>Phe</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td>34i</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>thr</td><td>will</td><td>Gly</td><td rowspan="2">asp</td><td>Leu</td><td>Ala</td><td>will</td><td>Leu</td><td rowspan="2">Arg</td><td>will</td><td>Ala</td><td>ile</td><td>asn</td><td rowspan="2">Arg</td><td>It's</td><td>will</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td> 365</td><td></td><td></td>

<td>glu</td><td>asn</td><td>Phe</td><td>will</td><td rowspan="2">asp</td><td>Phe</td><td>pro</td><td>Leu</td><td>Ile</td><td>pro</td><td>thr</td><td>glu</td><td>glu</td><td>glu</td><td>Leu</td><td>asn</td>

<td></td><td> 370</td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Arg</td><td>Gly</td><td>Gly</td><td rowspan="2">Arg</td><td>pro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 62 &lt;211&gt; 1170 &lt;212&gt; DNA &lt;213&gt; Listeria monocytogenes &lt;400&gt; 62

-144atgcgtgcga tgatggtagt tttcattact gccaactgca ttacgattaa ccccgacata atatttgcag cgacagatag cgaagattcc agtctaaaca cagatgaatg ggaagaagaa aaaacagaag agcagccaag cgaggtaaat acgggaccaa gatacgaaac tgcacgtgaa gtaagttcac gtgatattga ggaactagaa aaatcgaata aagtgaaaaa tacgaacaaa gcagacctaa tagcaatgtt gaaagcaaaa gcagagaaag gtccgaataa caataataac aacggtgagc aaacaggaaa tgtggctata aatgaagagg cttcaggagt cgaccgacca actctgcaag tggagcgtcg tcatccaggt ctgtcatcgg atagcgcagc ggaaattaaa aaaagaagaa aagccatagc gtcgtcggat agtgagcttg aaagccttac ttatccagat aaaccaacaa aagcaaataa gagaaaagtg gcgaaagagt cagttgtgga tgcttctgaa agtgacttag attctagcat gcagtcagca gacgagtcta caccacaacc tttaaaagca aatcaaaaac catttttccc taaagtattt aaaaaaataa aagatgcggg gaaatgggta cgtgataaaa tcgacgaaaa tcctgaagta aagaaagcga ttgttgataa aagtgcaggg ttaattgacc aattattaac caaaaagaaa agtgaagagg taaatgcttc ggacttcccg ccaccaccta cggatgaaga gttaagactt gctttgccag agacaccgat gcttctcggt tttaatgctc ctactccatc ggaaccgagc tcattcgaat ttccgccgcc acctacggat gaagagttaa gacttgcttt gccagagacg ccaatgcttc ttggttttaa tgctcctgct acatcggaac cgagctcatt cgaatttcca ccgcctccaa cagaagatga actagaaatt atgcgggaaa cagcaccttc gctagattct agttttacaa gcggggattt agctagtttg agaagtgcta ttaatcgcca tagcgaaaat ttctctgatt tcccactaat cccaacagaa gaagagttga acgggagagg cggtagacca &lt;210&gt; 63 &lt;211&gt; 123 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1170 &lt;400&gt; 63

-145-

<td rowspan="2">Met 1</td><td rowspan="2">Lys</td><td rowspan="2">Ala</td><td rowspan="2">val</td><td colspan="6">Leu Leu Leu Leu Ala Met</td><td colspan="2" rowspan="2">Ala Gly</td><td rowspan="2">Leu</td><td rowspan="2">Ala</td><td rowspan="2">Leu 15</td><td rowspan="2">gin</td>

<td colspan="2"> 5</td><td colspan="4"> 10</td>

<td>pro</td><td rowspan="2">Gly</td><td>thr</td><td>Ala</td><td>Leu</td><td>Leu</td><td rowspan="2">CYS</td><td rowspan="2">tyr</td><td>will</td><td rowspan="2">CYS</td><td rowspan="2">Lys</td><td>Ala</td><td>gin</td><td>val</td><td>will</td><td>asn</td>

<td></td><td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>glu</td><td>asp</td><td>CYS</td><td>Leu</td><td>gin</td><td>Val</td><td>glu</td><td>asn</td><td rowspan="2">CYS</td><td>thr</td><td>gin</td><td>Leu</td><td>Gly</td><td>glu</td><td>gin</td><td rowspan="2">CYS</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>trp</td><td>thr</td><td>Ala</td><td rowspan="2">Arg</td><td>ile</td><td rowspan="2">Arg</td><td>Ala</td><td>val</td><td rowspan="2">Gly</td><td>Leu</td><td>Leu</td><td>thr</td><td>val</td><td>ile</td><td>will</td><td rowspan="2">Lys</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Gly</td><td>CYS</td><td>will</td><td>Leu</td><td>asn</td><td>CYS</td><td>val</td><td rowspan="2">asp</td><td rowspan="2">asp</td><td>will</td><td>gin</td><td>asp</td><td rowspan="2">tyr</td><td rowspan="2">tyr</td><td>val</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Lys</td><td>Lys</td><td>asn</td><td>Ile</td><td>thr</td><td rowspan="2">CYS</td><td>CYS</td><td rowspan="2">asp</td><td>thr</td><td>asp</td><td>Leu</td><td>CYS</td><td>asn</td><td>Ala</td><td>will</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ala</td><td>His</td><td>Ala</td><td>Leu</td><td>gin</td><td>pro</td><td>Ala</td><td>Ala</td><td>Ala</td><td>ile</td><td>Leu</td><td>Ala</td><td>Leu</td><td>Leu</td><td>pro</td><td>Ala</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Leu</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu</td><td rowspan="2">trp</td><td rowspan="2">Gly</td><td>pro</td><td rowspan="2">Gly</td><td>gin</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 64 &lt;211&gt; 990 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (543) .. (543) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (580) .. (580) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature

-146 &lt;222&gt; (584) .. (584) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (604) .. (604) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (608) .. (608) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (615) .. (615) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (636) .. (636) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (640) .. (640) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (646) .. (646)

-147 &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (697) .. (697) &lt;223&gt; n oznacza a, c, g lub t &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (926) .. (926) &lt;223&gt; n oznacza a, c, g lub t &lt;400&gt; 64 agggagaggc tgcagccagg gcctgcaggt cagttggcct aggactacta gcggggccca tgctgctctg ggtgtggtgc cctggttcct ccnaaccctg tganacanat agcattttcc ννανν, νναι. the acaggcaatc acaagagttg ggggccaggc taataaacac agtgaccatg cactgccctg ggagaactgc cctgaccgtc cgtgggcaag tgccctgcag gggacccggc cccaggcctt gaggcacatc accttcccat ccgcntgcag acccttaacc &lt;-α uyaa and uya aggagggccc acgtgagttc ctcacatttg ctgttggata aaggctgtgc ctgtgctact acccagctgg atcagcaaag aagaacatca ccggctgccg cagctatagg tgtgccactc ctaacgcaag gggccttttc atggcccctc ctgtgttcag gccaggtttg agtaaaggct ctgggagttt tggggntccc agccaaaaaa tgcttgccct cctgcaaagc gggagcagtg gctgcagctt cgtgctgtga ccatccttgc ctctgggggg ctcacagaac tttgaccatg caggattccn caaccntttn gcacttnttc y ^Lv-'- y ^andyy *&gt; y gagatgaagt ccagagatgg gaatggcagc gttgatggca ccaggtgagc ctggaccgcg gaactgcgtg caccgacttg gctgctccct ccccgctgca ctggcccagt tatgtttgca accnggcaga tgttgntgtt ccccaggaag uCCCCCyCSC ggactgagta ggcctggagg ctgagcacag ggcttggccc aacgaggact cgcatccgcg gatgactcac tgcaacgcca gcactcggcc gcccacactg gggagcctgt ccccttttcc tcagttttag tccatggccc ccttccctgc ccagcagggg gaactggagg cctggaggaa cgtaggccct

120

180

240

300

360

420

480

540,.

‘600

660

720

7ΟΠ / UU₍

840

900

960

990

MIROSLAWA Ważyńska Rzecznik patentowy

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