Recombinant sendai virus

The present invention relates to recombinant Sendai virus and a method for producing the same.

Sendai virus is also called HVJ (Hemagglutinating virus of Japan) and is classified into Paramyxoviridae (Paramyxoviridae) and Paramyxovirus type 1 belonging to the genus Paramyxovirus.

Sendai virus particles are polymorphic, have an envelope with a diameter of 150 to 200 nm, and have genomic RNA (hereinafter referred to as "(-) strand RNA") that does not serve as a template for translation. Sendai virus is historically known as an industrially useful virus, and is particularly widely used for the production of cell heterokaryons and hybrid cells, that is, for cell fusion. It is also being developed as a material for membrane-fused liposomes and as a vector for gene therapy. Furthermore, Sendai virus is also used as an inducer for various interferons.

By morphological classification of genomic nucleic acids, Sendai virus belongs to the (-) single-strand RNA virus group of RNA viruses, (-) strand RNA viruses. RNA virus is dsRNA virus (double stranded RNA virus), (+) strand RNA virus And (-) strand RNA virus is classified into three types. The dsRNA virus group includes leovirus, rotavirus, plant reovir, etc., and has a plurality of segmental linear dsRNA genomes. (+) Strand RNA viruses include poliovirus, Sindbis virus, Semuliki forest virus, Japanese encephalitis virus, etc., and have one (+) strand RNA as a genome, and this RNA genome is simultaneously used as mRNA. It also functions and can produce proteins required for replication and particle formation depending on the translation function of the host cell. In other words, the genomic RNA of the (+) strand RNA virus itself has the power of transmission. In the present specification, "propagating power" means "infectious particles or a complex equivalent thereto after the nucleic acid existing in the cell is replicated after the nucleic acid is introduced into the cell by an infection or an artificial method. The ability to form and propagate to another cell one after another. " Sindbis virus, which is classified as (+) strand RNA virus, and Sendai virus, which is classified as (-) strand RNA virus, have infectivity and transmission ability, but are adeno-associated virus (Adeno) classified in the parvoviridae family. -associated virus) is infectious but not infectious (co-infection with adenovirus is required for the formation of virus particles). In addition, the (+) strand RNA derived from Sindbis virus artificially transcribed in vitro has a propagating power (forms virus particles when introduced into cells), but is artificially transcribed in vitro. Both the (+) and (-) strands of Sendai virus RNA have no transmissive power (even when introduced into cells, they do not form virus particles).

In recent years, virus-derived vectors have been used as vectors for gene therapy. In order to use the virus as a vector, it is necessary to establish a method for reconstructing virus particles. ("Reconstitution of virus particles" is the artificial production of nucleic acids in the viral genome to produce the original virus or recombinant virus in vitro or in cells.) Foreign genes This is because the viral particles must be recombined from the viral genome into which the exogenous gene has been incorporated by genetic manipulation in order to be introduced into the viral vector. If a virus reconstruction technique is established, it will be possible to produce a virus in which a desired foreign gene is introduced into the virus, a desired gene of the virus is deleted, or the virus is inactivated.

In addition, it is clear that if a viral reconstitution system is constructed and genetic manipulation of the virus becomes possible, it will be a great tool for genetically analyzing the function of the virus. Genetic analysis of viral function is extremely important from a medical point of view such as prevention and treatment of diseases. For example, if the replication mechanism of viral nucleic acid is elucidated, we will develop an antiviral agent with nucleic acid replication as the point of action, which has less damage to the host cell, by utilizing the difference from the nucleic acid replication mechanism in the host cell. It is possible to do. In addition, by elucidating the functions of proteins encoded by viral genes, it will be possible to develop antiviral agents targeting proteins involved in virus particle infectivity and virus particle formation ability. Further, by improving the gene related to the membrane fusion ability, it is expected that a more excellent membrane fusion liposome can be produced and used as a vector for gene therapy. In addition, as typified by interferon, infection with a virus activates a gene related to virus resistance of a host gene, and may exhibit virus resistance. Genetic analysis of viral function will also provide important insights into the activation of such host genes.

Reconstitution of DNA viruses using DNA as genomic nucleic acid has been carried out relatively early. For example, purified genomic DNA itself is used as monkeys, such as SV40 (J. Exp. Cell Res., 43,415-425 (1983)). It can be done by introducing it into the cells of.

The rearrangement of RNA viruses using RNA as genomic nucleic acid was preceded by the development of (+) strand RNA viruses. The reason for this is that genomic RNA also functions as mRNA. For example, in poliovirus, it was already reported in 1959 that the purified RNA itself has the ability to transmit (Journal of Experimental Medicine, 110, 65-89 (1959)). In addition, it has been reported that in Semliki forest virus (SFV), the virus can be reconstituted by introducing cDNA into the cells by utilizing the DNA-dependent RNA transcription activity of the host cell. (Journal of Virology, 65, 4107-4113 (1991)).

Furthermore, using these reconstruction techniques, the development of vectors for gene therapy is also underway [Bio / Technology, 11,916-920 (1993), Nucleic Acids Research, 23, 1495-1501 (1995), Human Gene. Therapy, 6,1161-1167 (1995), Methods in Cell Biology, 43,43-53 (1994), Methods in Cell Biology, 43,55-78 (1994)].

However, as mentioned above, although Sendai virus has many advantages that it can be used as an industrially useful virus, it is a (-) strand RNA virus, so a reconstitution system has not been established. It was. This is due to the extremely difficult viral particle reconstitution system via viral cDNA.

As described above, even if the RNA (vRNA; viral RNA) of the (-) strand RNA virus or its complementary strand RNA (cRNA; complementary RNA) is introduced into the cell alone, the (-) strand RNA virus may not be produced. It has been revealed. This is a decisive difference from the case of (+) strand RNA virus. Japanese Patent Application Laid-Open No. 4-211377 describes "a method for producing cDNA corresponding to the genome of a negative-strand RNA virus and an infectious negative-strand RNA virus", but the experimental contents of the publication are described as they are. "EMBO.J., 9,379-384 (1990)" has become clear that the experiment is not reproducible, and the author himself has completely withdrawn the content of the paper (EMBO.J., 10,3558 (1991). ) Therefore, it is clear that the technique described in JP-A-4-2111377 does not correspond to the prior art of the present invention.

(-) Regarding the rearrangement system of strand RNA virus, there is a report on influenza virus (Annu. Rev. Microbiol., 47, 765-790 (1993), Curr. Opin. Genet. DEV., 2, 77-81. (1992)). Influenza virus is a (-) strand RNA virus composed of an 8-segment genome. According to these reports, an exogenous gene was previously inserted into one of the cDNAs, and RNA transcribed from all eight cDNAs containing the exogenous gene was previously associated with a virus-derived NP protein to form an RNP. did. Reconstitution was established by supplying these RNPs and RNA-dependent RNA polymerase into the cells. Regarding the (-) positive-strand RNA virus, there is a report on the viral rearrangement from cDNA in the rabies virus belonging to the Rhabdoviridae family (J. Virol., 68, 713-719 (1994)).

Therefore, the (-) strand RNA virus reconstruction technology has basically become known, but in the case of Sendai virus, even if this method is applied as it is, the virus cannot be reconstructed. It was. In addition, the report that the virus particles were reconstituted in rhabdovirus was only confirmed by the expression of the marker gene, RT-PCR, etc., and could not be said to be sufficient in terms of production volume. Furthermore, conventionally, for the purpose of supplying the factors necessary for reconstitution in the cell, a virus such as a natural virus or a recombinant vaccinia virus is supplied to the cell at the same time as the nucleic acid of the virus to be reconstituted. However, there is a problem that it is not easy to separate the desired reconstructed virus from those harmful viruses.

<p> An object of the present invention is to establish a Sendai virus reconstitution system with high production efficiency, enable gene manipulation of Sendai virus, and supply a Sendai virus vector that can sufficiently withstand practical use in fields such as gene therapy.</p>

<p> The present inventors first first apply a cDNA or Sendai virus minigenome derived from Sendai virus DI particles (see protective interfering particle / EMBO.J., 10, 3079-3085 (1991)) for application to a reconstruction test of Sendai virus. Various studies were carried out using the cDNA of. As a result, the cDNA introduced into the cell, the cDNA group for transcriptional replication, and the recombinant vaccinia virus, which is a T7 RNA polymerase expression unit, We have found efficient conditions for the quantity ratio. The present inventors further obtain the full-length cDNA of Sendaivirus in both the (+) and (-) strands so that the (+) or (-) strand of Sendaivirus RNA is biosynthesized in the cell. A plasmid was constructed and introduced into the intracellular expression of the cDNA group related to transcriptional replication. As a result, we succeeded in reconstructing Sendai virus particles from Sendai virus cDNA for the first time. For efficient particle reconstruction, the present inventors prefer that the morphology of the cDNA introduced into the cell is circular rather than linear, and that (-) strand RNA is transcribed intracellularly. It was newly found that the efficiency of particle formation is higher when (+) strand RNA is transcribed intracellularly than when it is transcribed.</p><p> Furthermore, the present inventors have found that the Sendai virus can be reconstituted without using the recombinant vaccinia virus, which is a T7 RNA polymerase expression unit. That is, when the Sendai virus full-length RNA transcribed in vitro was introduced into cells and the cDNA of the early transcription replication enzyme group was transcribed under the control of the T7 promoter, the virus particles were reconstituted. This indicates that it is possible to produce recombinant Sendai virus without using any helper virus such as vaccinia virus by constructing cells expressing all of the early transcriptional replication enzyme groups. There is. Cells expressing all of the initial transcriptional replication enzyme groups are described in "J. Virology, 68,8413-8417 (1994)", and can be produced by those skilled in the art with reference to the description. .. The cells described in the literature are cells derived from 293 cells having NP, P / C, and L on the chromosome among the Sendai virus genes, and these are NP and P / C. , L expresses three proteins. </p><p> From the examples of many viral vectors, if the viral particles can be efficiently reconstituted from nucleic acids, the desired viral gene can be recombined, a foreign gene can be inserted, or the desired viral gene can be inactivated or missing. It is clear that the loss can be easily done by those skilled in the art. That is, it is obvious to those skilled in the art that the successful reconstruction of the Sendai virus particles for the first time in the present invention means that the genetic manipulation of the Sendai virus has become possible by the present invention.</p><p> That is, the present invention includes the following. (1) Recombinant Sendai virus that carries a genome containing the desired exogenous gene or in which the desired gene is deleted or inactivated and has the ability to transmit, (2) One or more functional protein genes are modified. The set according to (1) or (2), which comprises the recombinant Sendai virus according to (1), which is characterized by having an exogenous gene which can be expressed in a host (3). Substitute Sendai virus, RNA containing RNA contained in the recombinant Sendai virus according to any one of (4) (1) to (3), and any one of (5) (1) to (3). RNA containing cRNA of RNA contained in recombinant Sendai virus, DNA containing template cDNA capable of transcribing RNA according to (6) (a) (4) or (5), and (b) using the DNA as a template. As a kit containing a unit capable of transcribing the RNA described in (4) or (5) in vitro or in cells, (7) (a) Sendai virus NP protein, P / C protein and L protein (each protein). Is a kit containing a host expressing (which may be a protein having equivalent activity) and the RNA described in (b) (4) or (5), (8) Sendai virus NP protein, P / C protein and L. Described in any of (1) to (3), which comprises introducing the RNA according to (4) or (5) into a host expressing a protein (each protein may be a protein having equivalent activity). (9) (a) Host expressing Sendai virus NP protein, P / C protein and L protein, according to any one of (b) (4) or (5). DNA containing RNA or template cDNA capable of transcribing RNA, (c) a kit containing three of the units capable of transcribing RNA according to (4) or (5) in vitro or in cells using the DNA as a template. And (10) DNA containing a template cDNA capable of transcribing the RNA described in (4) or (5) into a host expressing the Sendai virus NP protein, P / C protein, and L protein, and in vitro or cells using the DNA as a template. The method for producing a recombinant Sendai virus according to any one of (1) to (3), which comprises introducing a unit capable of transcribing the RNA according to (4) or (5). ) A method for producing an exogenous protein, which comprises a step of infecting a host with the recombinant Sendai virus described in (3) and recovering the expressed exogenous protein, and (12) the recombinant Sendai virus described in (3). The orientation in which the culture or syrup containing the expressed exogenous protein, which can be obtained by introducing into the host and collecting the culture or syrup, and (13) the antisense RNA of the encoding protein are transcribed. DNA for expressing a protein encoded by the exogenous gene integrated into the Sendai virus vector, which comprises the exogenous gene located downstream of the promoter and the promoter.</p><p> In the recombinant Sendai virus vector of the present invention, for example, recombinant cDNA encoding the recombinant Sendai virus vector genome produced by genetic engineering is transcribed in vitro to produce recombinant Sendai virus genomic RNA. The RNA can be obtained by introducing the Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity) into a host that simultaneously expresses the RNA. Alternatively, the Sendai virus vector of the present invention uses (i) a recombinant cDNA encoding a genetically engineered recombinant Sendai virus vector genome, and (ii) an intracellular RNA using the DNA as a template. Transcriptable units can be obtained by introducing Sendai virus NP protein, P / C protein and L protein (each protein may be a protein having equivalent activity) into a host that simultaneously expresses it. In this case, for example, (i) can be DNA downstream of a particular promoter and (ii) can be DNA expressing a DNA-dependent RNA polymerase that acts on that particular promoter.</p><p> In the recombinant Sendai virus of the present invention, the Sendai virus used as a material before inserting a desired foreign gene or deleting or inactivating the desired gene is a strain classified into parainfluenza type 1. For example, Z strain (Sendai virus Z strain), Fushimi strain (Sendai virus Fushimi strain) and the like can be mentioned. Incomplete viruses such as DI particles and synthesized oligonucleotides can also be used as part of the material.</p><p> Further, in the recombinant Sendai virus of the present invention, as long as the transmissive power is maintained, any foreign gene is inserted in any site of RNA contained in the recombinant, and any genomic gene is deleted or deleted. It may be modified. Examples of the exogenous gene to be inserted include a gene encoding various cytokines and a gene encoding various peptide hormones that can be expressed in the host. To express the desired protein, an exogenous gene encoding the desired protein is inserted. In Sendai virus RNA, it is desirable to insert a sequence having a base number that is a multiple of 6 between the R1 sequence (5'-AGGGTCAAAGT-3') and the R2 sequence (5'-GTAAGAAAAA-3') ( Journal of Virology, Vol.67, No.8, (1993) p.4822-4830). Expression efficiency The expression level of the inserted foreign gene can be adjusted by the position of the gene insertion and the RNA base sequence before and after the gene. For example, in Sendai virus RNA, it is known that the closer the insertion position is to the NP gene, the higher the expression level of the inserted gene. The host for expressing the desired protein may be any cell infected with the recombinant Sendai virus, and examples thereof include cultured mammalian cells and chicken eggs. By infecting these hosts with a recombinant Sendai virus in which an expressible foreign gene is incorporated and recovering the expressed foreign gene product, the foreign gene product can be efficiently produced. The expressed protein can be recovered by a conventional method, for example, from a culture medium when a cultured cell is used as a host, or from a urinary serum when a chicken egg is used as a host.</p><p> When incorporating a foreign gene into a plasmid that biosynthesizes a (-) chain Sendai virus RNA, the foreign gene is promoted in the direction in which the antisense RNA of the protein encoded by the foreign gene is transcribed. Must be inserted downstream. Such a protein encoded by the exogenous gene integrated into the Sendai virus vector containing the exogenous gene arranged downstream of the promoter in the direction in which the antisense RNA of the encoding protein is transcribed and the promoter. "DNA for expression" was made available for the first time by the present invention and is a part of the present invention.</p><p> Further, for example, a gene involved in RNA replication of some Sendai viruses may be modified in order to inactivate a gene involved in immunogenicity or to increase the transcription efficiency and replication efficiency of RNA. Specifically, for example, at least one of NP protein, C / P protein, and L protein, which are replication factors, can be modified to enhance or weaken transcription and replication functions. In addition, HN protein, which is one of the structural proteins, has both hemagglutinin activity and neuraminidase activity, which are hemagglutinins. For example, if the former activity can be weakened, blood It may be possible to improve the stability of the virus in, and it is also possible to regulate the infectivity by, for example, modifying the activity of the latter. In addition, by modifying the F protein involved in membrane fusion, it can also be used to improve membrane fusion liposomes in which reconstituted Sendai virus and artificial liposomes containing desired drugs, genes, etc. are fused. is there.</p><p> The present invention has made it possible to introduce point mutations and insertions at arbitrary positions in genomic RNA, and it is highly expected that this will accelerate the accumulation of genetic knowledge of viral function. .. For example, if the replication mechanism of viral RNA is elucidated, we will develop an antiviral agent with nucleic acid replication as the point of action, which has less damage to the host cell, by utilizing the difference from the replication mechanism of nucleic acid derived from the host cell. It is possible to do. In addition, by elucidating the functions of proteins encoded by viral genes, it will be possible to develop antiviral agents targeting proteins involved in virus particle infectivity and virus particle formation ability. Specifically, for example, antigen-presenting epitopes of F protein and HN protein that can be antigen molecules on the cell surface. It can be used for analysis of data. In addition, when a gene related to virus resistance of a host gene is activated by infection with a virus and shows virus resistance, such activation of the host gene is also important by genetic analysis of virus function. Findings will be gained. Sendai virus is used in various basic experiments because it has an interferon-inducing effect. It is also conceivable to prepare a non-viral interferon inducer by analyzing the region required for this induction. The technique of the present invention can also be used for the development of vaccines. Live vaccines can also be produced by inoculating developed chicken eggs with a recombinant Sendai virus with artificially modified genes, and the findings obtained in this way can be used for other (-) strand RNA viruses, for example. , Measles virus, mumps virus-like vaccine It could also be applied to viruses that are in high demand. Furthermore, the present invention has also made it possible to use recombinant Sendai virus as a vector for gene therapy. Since the viral vector of the present invention is derived from Sendai virus, it is highly safe, and since the viral vector retains its transmissive power, it is expected that a large therapeutic effect can be obtained even with a small dose. When the treatment is completed and it becomes necessary to suppress the growth of the viral vector, or during the treatment, if an RNA-dependent RNA polymerase inhibitor is administered, only the growth of the viral vector is specific without damaging the host. Can be deterred.</p>

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] <u style="single">Preparation of Sendai virus transcription unit pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA</u> The plasmid pUC18 / T7 (-) HVJRz.DNA was prepared by inserting the T7 promoter, the Sendai virus cDNA designed to transcribe the (-) strand RNA, and the DNA carrying the ribozyme gene in this order into the pUC18 plasmid. In addition, a plasmid pUC18 / T7 (+) HVJRz.DNA was prepared by inserting the T7 promoter, Sendai virus cDNA designed to transcribe (+) strand RNA, and DNA carrying the ribozyme gene in this order into the pUC18 plasmid. did. The composition of pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA are shown in FIGS. 1 and 2.

[Example 2] <u style="single">Sendai virus reconstruction experiment from cDNA</u> 2,000,000 LLC-MK2 cells treated with normal trypsin and 2 ml of MEM medium (MEM + FBS 10%) were added to a plastic petri dish with a diameter of 6 cm, and CO was added.₂The cells were cultured for 24 hours under the conditions of 5% and 37 ° C. After removing the culture medium and washing with 1 ml of PBS, the recombinant vaccinia virus vTF7-3 expressing T7 polymerase prepared to have a multiplicity of infection of 2 was 0.1. Suspended in ml of PBS was added. Every 15 minutes, the petri dish was shaken so that the virus solution spread throughout, and the infection was performed for 1 hour. The virus solution was removed and washed with 1 ml PBS. A medium containing a cDNA solution was added to this petri dish. The medium containing the cDNA solution was prepared as follows.

The nucleic acids listed in the table (including plasmids expressing factors required for Sendai virus replication, pGEM-L, pGEM-P / C, pGEM-NP) were placed in a 1.5 ml sampling tube and HBS (Hepes buffered saline; 20 mM). Hepes pH 7.4, 150 mM NaCl) was added to bring the total volume to 0.1 ml. The (-) or (+) cDNA in the table indicates the plasmid pUC18 / T7 (-) HVJRz.DNA or pUC18 / T7 (+) HVJRz.DNA itself, with / C remaining cyclic and / L with the restriction enzyme MluI. It is shown that the cells are introduced into the cells after being linearized by.

On the other hand, 0.07 ml of HBS and 0.03 ml of DOTAP (manufactured by Boehringer Mannheim) were prepared in a polystyrene tube, and the nucleic acid solution was transferred to this polystyrene tube. In this state, it was allowed to stand for 10 minutes. To this was added cell culture medium (2 ml MEM + FBS 10%). Furthermore, rifampicin and cytosine arabinoside C / Ara C, which are vaccinia virus inhibitors, were added thereto so that the final concentrations were 0.1 mg / ml and 0.04 mg / ml, respectively. .. As a result, a medium containing the cDNA solution was prepared.

The above petri dish for 40 hours 5% CO₂ The cells were cultured under the conditions of 37 ° C. The cells in the petri dish were scraped off using a rubber policeman, transferred to an Eppendorf tube, centrifuged at 6,000 rpm for 5 minutes to precipitate only the cell components, and suspended again in 1 ml of PBS. A part of this cell fluid was inoculated into a 10-day-old embryonated chicken egg as it was or diluted. This cell solution was diluted with PBS to the cell count shown in Table 1, and 0.5 ml inoculated eggs were cultured at 35 ° C for 72 hours, then transferred to 4 ° C and left overnight. The serum urine solution of this egg was collected as a virus solution using a syringe and an injection needle.

The HAU (hemmaglutinin unit) and PFU (plaque forming unit) of the recovered virus solution were measured by the methods shown below.

The HAU was measured as follows. Chicken blood was centrifuged at 400 x g for 10 minutes and the supernatant was discarded. The remaining precipitate was suspended in 100 times the amount of PBS of the precipitate, centrifuged at 400 x g for 10 minutes, and the supernatant was discarded. This operation was repeated twice more to prepare a 0.1% blood cell solution. The virus solution was diluted 2-fold by serial dilution and 0.05 ml each was dispensed into a 96-well titer plate. A further 0.05 ml of blood cell solution was dispensed into this titer plate, vibrated lightly to mix well, and then allowed to stand at 4 ° C for 40 minutes. After that, the aggregation of erythrocytes was observed with the naked eye, and among the aggregated ones, the dilution rate of the one having the highest dilution rate of the virus solution was shown as HAU.

The PFU was measured as follows. CV-1 cells were grown in a monolayer on a 6-well culture plate. The culture plate medium was discarded, and 0.1 ml of the virus solution diluted 10-fold by the serial dilution method was dispensed into the wells in each culture plate and infected at 37 ° C. for 1 hour. Serum-free 2 × MEM and 2% agar were mixed at 55 ° C during infection, and trypsin was added to a final concentration of 0.0075 mg / ml. After 1 hour of infection, remove the virus solution and add 3 ml each of the medium mixed with agar to the wells in each culture plate, 5% CO₂It was kept warm at 37 ° C for 3 days under the conditions. 0.2 ml of 0.1% phenol red was added, and the mixture was kept warm at 37 ° C for 3 hours and then removed. The number of uncolored plaques was counted and the virus titer was evaluated as PFU / ml.

Table 1 shows the amount of Sendai virus cDNA used as a template introduced into LLC-MK2 cells, the amount of pGEM-L, pGEM-P / C and pGEM-NP, which are the cDNAs of factors required for RNA replication, incubation time, and chicken eggs. The number of inoculated cells, HAU, and PFU are shown respectively.<tables num="1"><img file="JP2005102702A_D0001.tif" /></tables> A sample showing both HAU and PFU was made into a sediment by ultracentrifugation, then resuspended and purified by 20% to 60% sucrose density gradient centrifugation, and the protein was separated by 12.5% SDS-PAGE. The protein contained was the same size as the protein of Sendai virus.

From this result, it was shown that cDNA can be introduced into cells to reconstitute Sendai virus. It was also shown that when the cDNA that transcribes the (+) strand is introduced into the cell, the virus particles are reconstructed more efficiently than when the cDNA that transcribes the (-) strand is introduced. Furthermore, it was shown that when the cDNA was introduced in a circular state, the virus particles were reconstituted more efficiently than when the cDNA was introduced in a linear form .

[Example 3] <u style="single">Examination of RNA replication factors required for Sendai virus rearrangement</u> An experiment was conducted to investigate whether all three need plasmids expressing L, P / C, and NP. The method is the same as in Example 2, but in Example 2, pGEM-L, pGEM-P / C, and pGEM-NP were introduced into cells together with cDNA, whereas in this experiment, pGEM- Only any two or one of L, pGEM-P / C and pGEM-NP was introduced into cells with cDNA.

Table 2 shows the amount of Sendai virus cDNA used as a template introduced into LLC-MK2 cells, the amount of pGEM-L, pGEM-P / C and pGEM-NP, which are the cDNAs of factors required for RNA replication, incubation time, and inoculation of chicken eggs. The number of cells, HAU, and PFU were shown respectively.<tables num="2"><img file="JP2005102702A_D0002.tif" /></tables> From Table 2, no virus production was observed when any combination of the two was introduced. As a result, it was confirmed that all three proteins are essential for reconstruction.

[Example 4] <u style="single">Sendai virus reconstruction experiment from in vitro transcribed RNA</u> In Example 2, it was shown that the Sendai virus was reconstituted from the cDNA, but it was further examined whether the same could be achieved with the products obtained by transcribing the cDNA in vitro, that is, vRNA and cRNA.

Sendai virus transcription unit pUC18 / T7 (-) HVJRz.DNA and pUC18 / T7 (+) HVJRz.DNA are linearized with the restriction enzyme MluI, and then purified T7 polymerase (EPICENTRE TECHNOLOGIES: Ampliscribe T7) is used as a template. In vitro RNA synthesis was performed using the Transcription Kit). The method of in vitro RNA synthesis followed the kit protocol. The RNA product obtained here was used in place of the cDNA of Example 2, and the same experiment was performed, and the virus production was evaluated by the HA test. The results are shown in Table 3.<tables num="3"><img file="JP2005102702A_D0003.tif" /></tables> From this result, the virus could be reconstructed regardless of which sense of RNA was introduced into the cell.

[Example 5]<u style="single">Examination of expression of foreign gene inserted in Sendai virus vector in host</u> (1) Preparation of Sendai virus vector "pSeVgp120" into which a foreign gene (HIV-1 gp120 gene) has been inserted Primer a (5'-TGCGGCCGCCGTACGGTGGCAATGAGTGAAGGAGAAGT-3') (SEQ ID NO: 1) and primer d (5'-TTGCGGCCGCGATGAACTTTCACCCTAAGT Using 3') (SEQ ID NO: 2), the HIV-1 gp120 gene on "pNI432" was amplified by standard PCR. TA cloning, digested with NotI, and digested with NotI "pSeV18"⁺I inserted it in. Next, this was transformed into E. Coli, the DNA of each colony of E. Coli was extracted by the "Miniprep" method, and electrophoresis was performed after DraIII digestion. A positive clone was obtained by selecting a clone confirmed to contain the DNA fragment of Escherichia coli (hereinafter, this positive clone is referred to as "clone 9"). After confirming that the nucleotide sequence was the target, DNA was purified by cesium chloride density gradient centrifugation. In addition, pSeV18 with gp120 inserted obtained by this⁺Is referred to as "pSeVgp120".

(2) Reconstitution of Sendai virus (SeVgp120) carrying pSeVgp120 and analysis of gp120 expression In the same manner as in Example 2 except that pSeVgp120 was further introduced into LLC MK2 cells in addition to pGEM NP, P, L. , The urine fluid of the developed chicken egg was collected, HAU was measured, and gp120 expression was examined (ELISA). The HAU was measured by the same method as in Example 2.

In addition, ELISA was performed as follows. A 100 μl sample was added to a 96-well plate covered with a monoclonal antibody against HIV-1 and reacted at 37 ° C for 60 minutes. After washing with PBS, 100 μl of HRP-binding anti-HIV-1 antibody was added, and the mixture was reacted at 37 ° C for 60 minutes. After washing this with PBS, tetramethylbenzidine was added, and the expression level of gp120 was measured by detecting the amount of the reaction product converted by HRP activity under acidic conditions with an absorbance of 450 nm. The results are shown on the left side of Table 4.

In addition, the obtained virus solution was infected with CV-1 cells, and the same examination was conducted. 5x10 CV-1 cells per plate⁵The cells were sown and grown, the medium was discarded, the cells were washed with PBS (-), a virus solution was added at an infection multiplicity of 10 and the cells were infected at room temperature for 1 hour. The virus solution was discarded, washed with PBS (-), plainMEM medium (MEM medium supplemented with antibiotics AraC, Rif and trypsin) was added, and the mixture was reacted at 37 ° C for 48 hours. After the reaction, the medium was collected, HAU was measured (the same method as in Example 2), and gp120 expression was examined (ELISA). The results are shown in the center of Table 4. The culture supernatant of CV-1 cells was inoculated again into the embryonated chicken eggs, and the HAU measurement results and the gp120 expression examination (ELISA) results of the virus solution obtained thereby are shown on the right side of Table 4.<tables num="4"><img file="JP2005102702A_D0004.tif" /></tables> As is clear from Table 4, a particularly high concentration of gp120 was produced in CV-1 cells (center of the table), and a high concentration of gp120 was also detected in the serous fluid inoculated into the embryonated chicken eggs again (right of the table). .. The results of 3 clones are shown on the left side of Table 4 and in the center of Table 4.

Furthermore, the expression of gp120 was analyzed by the Western blotting method. Medium of CV-1 cells infected with SeVgp120 is centrifuged at 20,000 rpm for 1 hour to precipitate the virus, and the supernatant is subjected to TCA (10% (v / v), 15 minutes on ice) or 70% ethanol ( Treat at -20 ° C), centrifuge at 15,000 rpm for 15 minutes, mix the precipitated protein with "SDS-PAGE Sample buffer" (Daiichi Kagaku) and react at 90 ° C for 3 minutes on a 10% acrylamide gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After the electrophoresis, the protein was transferred to a PVDF membrane (first chemistry), and the monoclonal antibody 902 was reacted at room temperature for 1 hour. Then, the cells were washed with T-TBS, anti-mIgG (Amersham) was reacted at room temperature for 1 hour, and the cells were washed with T-TBS. Furthermore, HRP-binding protein A (Amersham) was reacted at room temperature for 1 hour and washed with T-TBS. 4-Chloro-1-naphthol (4CNPlus) (Daiichi Kagaku) was added to this, and gp120 was detected. As a result, a band was detected at the position of the expected molecular weight of gp120.

Furthermore, the relationship between the time after infection of CV-1 cells with SeVgp120, the HAU value, and the expression level of gp120 was analyzed. 5x10 on a 10cm plate⁶CV-1 cells are sown to form cells, infected with SeVgp120 at a multiplicity of infection of 10, then 1 ml of medium is collected at 30,43,53,70 hours and mixed with an equal volume of fresh medium. HAU was measured, gp120 expression was examined (ELISA) and western blotting was performed. The result is shown in Fig. 3. As is clear from Fig. 3, the production of gp120 tended to increase as the HAtiter of Sendai virus increased.

[Example 6]<u style="single">Analysis of SeVgp120 proliferation and gp120 expression in various types of cells</u> HAU was measured and gp120 expression was examined (ELISA) in the same manner as in Example 5 except that various types of cells were used. The results are shown in Table 5.<tables num="5"><img file="JP2005102702A_D0005.tif" /></tables> The left side of the table shows the time after infection of various types of cells with SeVgp120. As a result, proliferation of SeVgp120 and expression of gp120 were detected in all the cells examined.

[Example 7]<u style="single">Examination of expression of luciferase gene inserted in Sendai virus vector in host</u> Primer (5'-AAGCGGCCGCCAAAGTTCACGATGGAAGAC-3'(30mer)) (SEQ ID NO: 3) and primer (5'-TGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGATTATTACAATTTGGACTTTCCGCCC-3' (69mer)) (SEQ ID NO: 4) to isolate the luciferase gene for vector insertion. ), And using "pHvluciRT4" as a template, a luciferase gene with NotI sites added to both ends was isolated by a standard PCR method. Then this was digested with NotI pSeV18⁺The Sendai virus vector into which the luciferase gene was inserted was obtained. It was then introduced into LLCMK2 cells and inoculated into developing chicken eggs. The allantois membrane of the developing egg was cut off, washed twice with cold PBS (-), 25 μl of lysis buffer (Picagene WACO) was added, and the mixture was stirred well and then centrifuged at 15000 rpm for 2 minutes. 5 μl of the supernatant was collected, 50 μl of substrate (IATRON) was added, the mixture was placed in a 96-well plate, and the fluorescence intensity was measured with a luminous meter (Luminous CT-9000D, DIA-IATRON). The activity was expressed in cps (counts per second). As a result, particularly high luciferase activity was detected in CV-1 cells 24 hours after infection (Table 6). In addition, Sendai virus in which the luciferase gene was not introduced was used as a control (indicated by "SeV" in the table). In addition, the table shows the detection results of 2 clones.<tables num="6"><img file="JP2005102702A_D0006.tif" /></tables>

INDUSTRIAL APPLICABILITY The present invention establishes a system for recombining virus particles more efficiently than Sendaivirus cDNA, enables gene manipulation in Sendaivirus, and contains a genome containing a desired exogenous gene or in which a desired gene is deleted or inactivated. It has become possible to obtain a recombinant Sendai virus that retains the virus and has a transmitting power.

<figref num="1">FIG. 1 is a diagram showing the composition of pUC18 / T7 (+) HVJRz.DNA.</figref><figref num="2">FIG. 2 is a diagram showing the composition of pUC18 / T7 (-) HVJRz.DNA.</figref><figref num="3">FIG. 3 is a diagram showing the relationship between the time after infection of CV-1 cells with SeVgp120, the HAU value, and the expression level of gp120.</figref>