Human papillomavirus nucleic acid hybridization probes and methods for employing the same.

Description: TECHNICAL FIELD [Detailed description of the invention]

[0001] The present invention relates to the types of human papillomavirus, particularly human papillomavirus type 35 (hereinafter "HPV 35"), human papillomavirus type 43 (hereinafter "HPV 43"), and human papillomavirus. It relates to a nucleic acid hybridization probe for type 44 (hereinafter "HPV 44") and type C57 of human papillomavirus (hereinafter "HPV C57"), and a method using the same.

(A) Types of human papillomavirus Human papillomavirus (hereinafter "HVP") has been identified as the cause of various epithelial lesions, such as warts, condyloma and dysplasia [see, Gissman, L .; , Cancer Surv., 3: 161 (1984); Pfister, H. et al. Biochem. Pharmacol., 99: 111 (1983); Durst, M. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 3812 (1983) and Boshart, M. et al. Et al., EMBO J. 3: 1151 (1984)]. Cervical dysplasia [also known as cervical intraepithelial dysplasia (CIN)] progresses to cancer of the cervix; mild dysplasia (CIN I) to moderate dysplasia (CIN) II), severe dysplasia, in-situ cancer (collectively CIN III), and progression to invasive cancer are believed to be early events.

Studies examining the association of HPV types with cervical dysplasia and cervical cancer have shown that HPV types 6, 11, 16, 18, 31 and 33 are associated with genital lesions. See, Gissman, L., Cancer Surv., 3: 161 (1984); Pfister, H. et al. Biochem. Pharmacol., 99: 111 (1983); Durst, M. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 3812 (1983); Boshart, M. et al. Et al., EMBO J. et al. 3: 1151 (1984); de Villiers (de) Villiers), E.I. -M. Et al., Journal of Virology (J. Virol.), 40: 932 (1981); Gissman, L. et al. Et al., Journal of Virology (J. Virol.), 58: 225 (1986) and Beaudenon, S. et al. , Nature, 321 and 246 (1986)].

HPVs are classified into types based on their DNA sequence similarity. Two HPVs are taxonomically classified as the same type if their DNA cross-hybridizes more than 50%. The measurement was performed by hybridization in solution under moderately stringent hybridization conditions, followed by separation of the double-stranded DNA from the DNA by chromatography of hydroxyapatite, the conditions being complete. It is defined as approximately 25 ° C lower than the melting point of base paired double-stranded DNA (conveniently described as Tm-25 ° C). The melting point (Tm) of a fully base paired double-stranded DNA can be accurately predicted using the following well-established formula: Tm = 16.6 × log [Na⁺] +0.4 1 ×% G: C + 81.5-0.72 × (%) (v / v) Formaldehyde The above formula experimentally measures Tm for each individual DNA under each hybridization condition. A convenient means of setting acid focus to determine non-stringent and stringent hybridization conditions for various DNAs in solutions with varying salt and formaldehyde concentrations without the need to do so. provide.

Less than 50% of each HPV DNA is cross-hybridized in solution under moderate stringent conditions, measured and defined by its ability to bind hydroxyapatite, completely or partially. HPV DNAs are not sufficiently relevant for taxonomic separation to be of the same type if they are capable of forming double-stranded structures. A 50% cross-hybridization cutoff using this method is used as a consensus criterion for the assignment of new HPV types for naming purposes. This method of measuring the degree of cross-hybridization between HPV DNAs is used to determine whether two HPV DNAs represent a common type of different isolate or a different type of isolate. It has been historically applied as a method of hybridization. The use of this criterion precedes the establishment of clinical criteria for determining and defining the type of HPV. As detailed below, the clinical criteria (gevifal) for determining and defining the type of HPV are based on the epidemiological distribution of the type of HPV among genital lesions.

The aforementioned method of measuring the degree of cross-hybridization is based on assessing the degree of formation of double-stranded DNA molecules completely or partially after the hybridization reaction. However, it should be noted that conversion of 50% of DNA to a double-stranded DNA molecule, completely or partially, does not mean that the nucleotide sequences of the DNA are 50% homologous.

As mentioned above, HPV can also be divided into types based on clinical criteria. That is, it has been observed that different types of HPV exhibit a clear epidemiological distribution between genital lesions of different severity and between different constitutive populations, as defined by the aforementioned cross-hybridization criteria. It was.

For example, HVP 6 and HVP 11 are primarily associated with amphoteric lesions, such as outwardly growing condyloma and, to a lesser extent, condyloma [see, Gissman, L. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 560 (1983)]. HVP 6 and HVP 11 are also detected in certain rare types of malignant epithelial cancers [see Zachow, K. et al. R. Et al., Virology Journal (J. Virol.), 57: 353 (1986)]. In contrast, HVP 16, HVP 31 and HVP 33 are detected at varying frequencies in cervical and other anogenital cancers and their precursor lesions [Reference, Durst, M. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 3812 (1983), Boshart, M.D. Et al., EMBO J. et al. 3: 115 (1984), Lorincz, A. et al. T. Et al., Journal of Virology (J. Virol.), 58: 225 (1986) and Beaudenon, S. et al. , Nature, 321 and 246 (1986)]. This distribution of HVP 16, HVP 18, HVP 31 and HVP 33 shows HVP 16, HVP 18, HVP 31 and HVP compared to lesions infected with HVP 6 and HVP 11. It is believed to reflect the greater risk or more rapid progression of cervical cancer infected at 33. After all, determining the type of HPV has clinical-diagnostic value. That is, it is an important factor in assessing the risk of developing cancer in patients who show evidence of HPV infection. Appropriate therapeutic treatment can be selected based on the assessed risk of developing cancer.

In addition, HVP 16 has been widely disseminated in Europe than in Africa [Durst, M. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 3812 (1983)], whereas HVP 18 is more widespread in Africa than in Europe [Boschart]. (Boshart), M.D. Et al., EMBO J. et al. 3: 1115 (1984)].

Thus, within the relevant scope of the invention, the two HPVs are (1) if they meet the criteria for the degree of cross-hybridization described above, or (2) they are between genital lesions. Both show substantially the same epidemiological distribution of cross-hybridization and are considered to be of the same type when cross-hybridizing with the same genital lesions constituting the epidemiological distribution.

It has been found that a significant percentage of cervical cancers and genital lesions that may progress to cervical cancers contain "new" HPV types that do not correspond to any of the known HPV types. It was. Thus, these "new" HPV types can be detected and classified in the light of the known association between specific HPV types and genital lesions at high risk of developing cervical cancer. Can confirm the risk of cervical cancer associated with these "new" HPV types in patients who have evidence of HPV infection and can be infected with these "new" HPV types.

(B) Cloning of HPV types Despite long stagnant efforts in the field, it has been impossible to grow HPV in cell culture in vitro. However, recombinant DNA cloning techniques have made it possible to separate and purify the DNA of many HPV types, such as HPV types 6, 11, 16, 18, 31 and 33 [see Durst (Reference, Durst). Durst), M.D. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80: 3812 (1983), Boshart, M.D. Et al., EMBO J. et al. 3: 1151 (1984), de Villiers, E.I. -M. Et al., Journal of Virology (J. Virol.), 40: 932 (1981), Gissman, L. et al. Et al., Journal of Virology (J. Virol.), 58: 225 (1986), Lorincz, A. et al. T. Et al., Virology Journal (J. Virol. ), 58: 225 (1986), and Beaudenon, S. et al. , Nature, 321 and 246 (1986)]. Most of the knowledge about HPV uses these DNA sequences to prepare nucleic acid hybridization probes for studying DNA sequences in such recombinant DNA and detecting HPV in tissue samples. It was induced by that.

(C) Hybridization probe As mentioned above, HPV DNA was used as a hybridization probe for different types of HPV. Two different types of HPV DNA can be easily distinguished by hybridization under stringent hybridization conditions using such hybridization probes, and stringent hybridization conditions are fully base paired. It is defined as approximately 10 ° C. lower than the melting point of the double-stranded DNA hybrid (conveniently described as Tm-10 ° C.). Similarly, it can be easily distinguished by hybridization under stringent hybridization conditions using such hybridization probes, and stringent hybridization conditions are fully base paired double-stranded DNA-. It is defined as approximately 10 ° C lower than the melting point of RNA hybrids (conveniently described as Tm-10 ° C). In addition, two different types of HPV RNA can be easily distinguished by hybridization under stringent hybridization conditions using such hybridization probes, and stringent hybridization conditions are fully base paired double-stranded RNA-. It is defined as approximately 10 ° C lower than the melting point of RNA hybrids (conveniently described as Tm-10 ° C). It should be noted that HPV DNA or RNA displayed as different types using the above criteria can, in fact, be as much as 80% of their nucleotide sequences in common.

In addition, two different types of HPV DNA can be cross-hybridized to non-stringent hybridization conditions using such hybridization probes, and non-stringent hybridization conditions are fully hybridized. It is defined as approximately 35 ° C. or higher (conveniently described as Tm-35 ° C. or higher) below the melting point of a base paired double-stranded DNA-DNA hybrid. Similarly, one type of HPV DNA uses such a hybridization probe to bring other types of HPV to non-stringent hybridization conditions. Hybridization conditions that allow cross-hybridization with RNA and are not stringent are approximately 35 ° C or higher below the melting point of fully base paired double-stranded DNA-RNA hybrids (conveniently described as Tm-35 ° C or higher). Is defined as. In addition, two different types of HPVRNA can be cross-hybridized to non-stringent hybridization conditions using such hybridization probes, and non-stringent hybridization conditions are fully base paired. It is defined as approximately 35 ° C or higher below the melting point of double-stranded RNA-RNA hybrids (conveniently described as Tm-35 ° C or higher). [See, Anderson, L .; M. Et al., Nucleic Acid Hybridization, pp. 73-111, B. et al. D. Hames and S.M. J. Higgins ed., I. R. L. Press, Oxford, United Kingdom and Washington, D.C. C. , United States (1985)].

The melting points of DNA-DNA, DNA-RNA and RNA-RNA of the same nucleotide sequence can vary in different chemical environments. The effects of various compounds on the associated melting points of these various hybrids have been studied for several reagents. For example, increasing the concentration of formaldehyde makes DNA-DNA hybrids differentially more destabilized than DNA-RNA, so at high concentrations of formaldehyde, eg, 80% (v / v), DNA-RNA is the same nucleotide. It is well known that it can have a significantly higher melting point than a DNA-DNA hybrid of a sequence.

As mentioned above, the melting points of DNA-DNA hybrids are determined by Anderson, L. et al. M. Et al., Nucleic Acid Hybridization, pp. 73-111, B. et al. D. Hames and S.M. J. Higgins ed., I. R. L. Press, Oxford, United Kingdom and Washington, D.C. C. , The United States (1985), as described in Y, can be predicted. In addition, the melting points of DNA-DNA hybrids are determined by Howley, P. et al. Et al., As described in the Journal of Biochemistry (J. Biochem.), 254: 4876 (1979), can be determined experimentally. The melting point of DNA-RNA hybrids can also be determined by means well known in the art.

Thus, tissue samples are generally high for the presence of HPV DNA or RNA and / or in particular HPV DNA or RNA types, i.e. stringent or non-stringent hybridization conditions. Depending on whether it is used for hybridization, it can be tested by hybridization of nucleic acids.

Therefore, one object of the present invention is to establish and contain whether genital lesions that may progress to cervical cancer and cervical cancer contain a "new" type of HPV. The case is to clone the hypothesized "new" HPV type.

Another object of the present invention is to provide nucleic acid hybridization probes that are generally specific for HPV types and especially for "novel" HPV types.

Yet another object of the present invention is generally HPV DNA or RNA and particularly "novel" in unknown samples of DNA or RNA, particularly DNA or RNA derived from genital lesions. It is to provide a method of detecting DNA or RNA of HPV to determine the risk of developing cervical cancer.

These and other objectives of the invention will be apparent from the following detailed description of the invention.

In the present invention, the types of "new" HPV cloned in the present invention have been found to be novel HPV types, which are labeled HPV 35, HPV 43, HPV 44 and HPV C 57.

Thus, in one embodiment, the aforementioned object of the invention is the cloning vector and substantially all of HPV 35 DNA or fragments thereof, substantially all of HPV 43 DNA or fragments thereof, HPV, respectively. Satisfied with recombinant DNA of HPV 35, HPV 43, HPV 44 or HPV C57, which comprises substantially all of 44 DNA or fragments thereof, or substantially all of HPV C57 DNA or fragments thereof. ..

In other embodiments, the aforementioned object of the invention is essentially pure HPV 35 DNA or a fragment thereof, essentially pure HPV 43 DNA or a fragment thereof, essentially pure HPV 44 DNA or a fragment thereof. Fragments, or essentially pure HPV C57 DNA or fragments thereof; and essentially pure HPV 35 RNA or fragments thereof, essentially pure HPV 43 RNA or fragments thereof, essentially pure HPV 44 RNA or fragments thereof , Or essentially pure HPV C57 RNA or fragments thereof.

In still other embodiments, the aforementioned objects of the invention are: (1) under non-stringent conditions, (a) (i) marker-labeled HPV 35 DNA or fragments thereof, markers. Labeled HPV 43 DNA or fragments thereof, marker-labeled HPV 44 DNA or fragments thereof, or marker-labeled HPVC 57 DNA or fragments thereof, and (ii) marker-labeled HPV 35 RNA or fragments thereof, A member selected from the group consisting of marker-labeled HPV 43 RNA or fragment thereof, marker-labeled HPV 44 RNA or fragment thereof, or marker-labeled HPV C57 RNA or fragment thereof, and (b). ) Perform hybridization using an unknown sample of DNA or RNA, and (2) assay for the presence of cross-hybridation to detect DNA or RNA of HVP in said sample. It was satisfied by the method of detecting DNA or RNA of HVP characterized by the above.

In other embodiments, the aforementioned objects of the invention are: (1) under stringent conditions, (a) and (i) marker-labeled HPV 35 DNA or fragments thereof, markers, respectively. HPV 43 DNA or fragment labeled with, marker-labeled HPV 44 DNA or fragment thereof, or HPV C57 DNA labeled with marker or fragment thereof, and (ii) marker-labeled HPV 35 RNA, respectively. Or a member selected from the group consisting of a fragment thereof, a marker-labeled HPV 43 RNA or a fragment thereof, a marker-labeled HPV 44 RNA or a fragment thereof, or a marker-labeled HPV C57 RNA or a fragment thereof. , And (b) an unknown sample of DNA or RNA, and (2) assayed for the presence of cross-hybridation, respectively, in HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 Detected by a method of detecting HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPVC 57 DNA or RNA, which comprises detecting DNA or RNA.

In still other embodiments, the aforementioned objects of the invention are: (1) under stringent conditions, (a) DNA or RNA derived from each genital lesion in sampling of genital lesions. The first fraction of, said sampling indicates epidemiological progression to cervical cancer, and (b) (i), marker-labeled HPV 35 DNA or fragments thereof, marker-labeled HPV 43 DNA, respectively. Or a fragment thereof, a marker-labeled HPV 44 DNA or a fragment thereof, or a marker-labeled HPV C57 DNA or a fragment thereof, and (ii) a marker-labeled HPV 35 RNA or a fragment thereof, labeled with a marker, respectively. Hybridization using a member selected from the group consisting of HPV 43 RNA or fragments thereof, marker-labeled HPV 44 RNA or fragments thereof, or marker-labeled HPV C57 RNA or fragments thereof. And (2) under stringent conditions, (a) a second fraction of DNA or RNA derived from each genital lesion in the sampling of the genital lesion, and Hybridization was performed using (b) an unknown sample of DNA or RNA derived from marker-labeled genital lesions, and (3) epidemics of cross-hybridation obtained in step (1). HPV 35 DNA or RNA, HPV 43 DNA in the sample, respectively, comparing the target distribution with that obtained in step (2) and cross-hybridation of DNA from each lesion constituting the epidemiological distribution. Alternatively, it comprises detecting RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPVC 57 DNA or. Satisfied with the method of detecting RNA.

Conventionally unknown types of HPV have been discovered in the present invention and are labeled HPV 35, HPV 43, HPV 44 and HPV C 57. HPV 35, HPV 43, HPV 44 and HPV C57 were first cloned in the present invention and thus of DNA or RNA generally derived from unknown samples of DNA or RNA, especially HPV DNA or RNA and especially genital lesions . In unknown samples, it is generally possible to prepare hybridization probes for the detection of HPV DNA or RNA, in particular HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively. It became.

HPV 35 is described in Washington, D.C. C. It was isolated and cloned from the adenocarcinoma biopsy obtained in.

HPV 43 was isolated and cloned from a biopsy of vulvar tissue obtained from Michigan, showing only hyperkeratosis on histopathological examination.

HPV 44 was isolated and cloned from a biopsy of vulvar condyloma from Michigan.

HPV C57 is available in Washington, D.C. C. It was isolated and cloned from a biopsy of the vulvar condyloma obtained from.

First, HPV 35, HPV 43, HPV 44 and HPV C57 were cloned to clone HPV 35 clones 1A and 1B, HPV 43 clones 1A and 1B, HPV 44 clone 1 and HPV C57 clones 1A and 1B. The particular cloning vector used in the examples provided here to prepare was λ L47.

HPV 35 DNA from clones 1A and 1B of HPV 35 was subcloned into pBR322 (ATCC No. 37017) to prepare clones 2A and 2B of HPV 35. HPV 35 clones 2A and 2B are from the American Type Culture Collection, respectively, at ATCC No. It was commissioned at 40330 and 40331.

HPV 43 DNA from clones 1A and 1B of HPV 43 was subcloned into pT713 [GIBCO / BRL, Gaithersburg, Mariyland] to prepare clones 2A and 2B of HPV 43. HPV 43 clones 2A and 2B are from the American Type Culture Collection, respectively, at ATCC No. It was commissioned at 40338 and 40339.

HPV 44 DNA from clone 1 of HPV 44 was subcloned into pT713 [GIBCO / BRL, Gaithersburg, Calif.] To prepare clone 2 of HPV 44. Clone 2 of HPV 44 was presented at ATCC No. in the American Type Culture Collection. It was commissioned at 40353.

HPV C57 DNA from clones 1A and 1B of HPV C57 was subcloned into pT713 [GIBCO / BRL, Gaithersburg, Mariyland] to prepare clones 2A and 2B of HPV C57. HPV C57 clones 2A and 2B are from the American Type Culture Collection, respectively, at ATCC No. It was commissioned at 40341 and 40379.

HPV 35 DNA, in its entirety, is excised from HPV 35 clones 2A and 2B using the BamHI restriction endonuclease and subcloned into any well-known prokaryotic and eukaryotic cloning vector. I was able to do it.

Overall, HPV 43 DNA was excised from clone 2A of HPV 43 using the HindIII restriction endonuclease and from clone 2B using the BamHI restriction endonuclease, and any well-known prokaryote and It could be subcloned in a prokaryotic cloning vector.

Overall, HPV 44 DNA can be excised from clone 2 of HPV 44 using the BamHI restriction endonuclease and subcloned into any well-known prokaryotic and eukaryotic cloning vector. It was.

HPV C57 DNA is excised in its entirety using EcoRI-restricted endonuclease from clone 2A of HPV C57 and using BamHI-restricted endonuclease from clone 2B, and any well-known prokaryote and It could be subcloned in a prokaryotic cloning vector.

The particular cloning vector used for subcloning HPV 35, HPV 43, HPV 44 or HPV C57 is non-critical and any known prokaryotic cloning vector such as pUC11, λ induction vector, eg. , Λ Sharon or M13-induced bacteriophage [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, New York, Cold Spring Harbor (1982) and Loenen. , W. A. M. Et al., Gene, 20: 249 (1980)] or any known eukaryotic cloning vector, such as pZIP-Neo SV [X1] or pBKTK-1 [see, Poueels, P. et al. H. Et al., Cloning Vectors: Laboratory Manual (A Laboratory) Manual), Elseiver, Amsterdam (1985)].

The fragments of HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA are similarly clones 2A and 2B of HPV 35, clones 2A and 2B of HPV 43, clone 2 of HPV 44, or HPV 44, respectively. HPV C57 clones 2A and 2B can be excised using other well-known restriction endonucleases and cloned in the cloning vector described above. Similarly, HPV 35 DNA in clones 2A and 2B of HPV 35, HPV 43 DNA in clones 2A and 2B of HPV 43, HPV 44 DNA in clone 2 of HPV 44, or in clones 2A and 2B of HPV C57. HPV C57 DNA was excised from them, bound together, and cloned in the cloning vector described above to substantially the genome of HPV 35, the genome of HPV 43, the genome of HPV 44 and the genome of HPV C57, respectively. A vector containing all of them can be obtained.

Cloning of HPV 35 DNA or fragments thereof, HPV 43 DNA or fragments thereof, HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof is generally HPV DNA or RNA, particularly HPV 35 DNA or RNA, respectively. , HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively, to prepare large amounts of HPV 35 DNA or fragments thereof, HPV 43 DNA or fragments thereof, respectively. , HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof can be produced relatively easily.

In addition, HPV 35 DNA or fragments thereof, HPV 43 DNA or fragments thereof, HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof are subcloned and identified in other well-known cloning vectors. The advantage of obtaining the special properties of the cloning vector of is available, and these properties are homologous to HPV 35 DNA, HPV 43 DNA, HPV 44 DNA, or HPV C57 DNA inserted in the cloning vector. Promotes in vitro synthesis of sex RNA [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982)]. Examples of these cloning vectors include pT712 and pT713, each of which is commercially available from GIBCO / BRL, Gaithersburg, Maryland. HPV 35 DNA or fragments thereof, HPV 43 DNA or fragments thereof, HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof can be subcloned into these cloning vectors, so that HPV 35 DNA or fragments thereof, respectively. Fragments, HPV 43 DNA or fragments thereof, HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof serve as efficient templates for phage-encoded RNA polymerases such as T7, T3 or SP6. Can be done. Using such cloning vectors and such RNA polymerases, HPV 35 DNA or fragments thereof, HPV 43 DNA or fragments thereof, HPV, respectively. HPV 35 RNA, HPV 43 RNA, HPV44 RNA, or HPV C57 RNA complementary to any of 44 DNA or fragments thereof, or HPV C57 DNA or strands of fragments thereof, using methods well known in the art. It can be synthesized by in vitro transcription.

A particular bacterium or eukaryotic host for propagating a cloning vector containing HPV 35 DNA or a fragment thereof, HPV 43 DNA or a fragment thereof, HPV 44 DNA or a fragment thereof, or HPV C57 DNA or a fragment thereof. It will depend on the cloning vector used. For example, a typical host for propagating HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA cloned in λ L47 is E. coli. coli NM538 [Frinschanf, A.M. M. Et al., Journal of Molecular Biology (J. Mol. Biol.), 170: 827 (1983)]. Other hosts, such as E. coli. coli HB101 [Boyer, H. et al. W. Et al., Journal of Molecular Biology (J. Mol. Biol.), 41: 459 (1969)] can be used when using pBR322 or pUC11 as the cloning vector. HPV 35 DNA, HPV cloned in pZIP-Neo SV [X1] A typical host for growing 43 DNA, HPV 44 DNA or HPV C57 DNA is a monkey Cos cell, whereas a typical host for growing HPV DNA cloned in pBKTK-1 is. It may be found in one of a number of well-known mammalian cell lines [eg, Poueels, P. et al. H. Et al., Cloning Vectors: Laboratory Manual (A Laboratory Manual, Elseiver, Amsterdam (1985)].

Hybridization of the probe of the present invention to HPV DNA or RNA or particularly HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA is a hybridization condition used. Will depend on. That is, under non-stringent hybridization conditions, HPV 35 DNA or RNA or fragments thereof, HPV 43 DNA or RNA or fragments thereof, HPV 44 DNA or RNA or fragments thereof, or HPV C57 DNA or RNA or fragments thereof are generally used. It can be used as a hybridization probe for HPV DNA or RNA. On the other hand, under stringent hybridization conditions, HPV 35 DNA or RNA or fragments thereof, HPV 43 DNA or RNA or fragments thereof, HPV 44 DNA or RNA or fragments thereof, or HPV C57 DNA or RNA or fragments thereof In particular, HPV35 DNA or RNA, HPV 43, respectively. It can be used as a hybridization probe for DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA.

As mentioned above, different types of HPV DNA and RNA are fully base paired under non-stringent hybridization conditions, i.e., having a base composition equal to that of HPV DNA or RNA as a general group. Cross-hybridization is possible at temperatures approximately 35 ° C. or higher below the melting point of double-stranded DNA.

In addition, unknown samples of DNA or RNA are fully base paired double-stranded DNA under stringent hybridization conditions, i.e., having a base composition equivalent to that of HPV DNA or RNA as a general group. By performing cross-hybridization at a temperature approximately 10 ° C. below the melting point of, it is possible to test for the presence of a particular type of HPV and identify that type.

In the method of the present invention, hybridization under non-stringent hybridization conditions is carried out by first hybridization under non-stringent hybridization conditions and then washing under non-stringent hybridization conditions. ..

Further, in the method of the present invention, hybridization under stringent hybridization conditions is performed by first hybridization under non-stringent hybridization conditions and then washing under stringent hybridization conditions. Alternatively, it is carried out by hybridization under stringent hybridization conditions and then washing under stringent hybridization conditions. In the first method, ie, hybridization under non-stringent hybridization conditions and then washing under stringent hybridization conditions, the hybrids that form between different types of DNA or RNA are unstable. Hybrids that form between the same type of DNA or RNA are stable.

[0052] Hybridization is preferably performed under non-stringent hybridization conditions to determine if an unknown sample of DNA or RNA has the same or different HPV type as the hybridization probe used, followed by stringin. Wash under non-gent hybridization conditions. After assaying for the presence of hybrids, the detected hybrids are washed under stringent hybridization conditions. In this method, the amount of hybrids remaining after hybridization under non-stringent hybridization conditions is determined and compared to the amount of hybrids present after washing under stringent hybridization conditions. If the amount of hybrids formed by washing under stringent hybridization conditions is little or minimally reduced, then this is the hybrid that was originally formed, i.e., the hybrid that was formed under non-stringent hybridization conditions. Indicates that they were of the same type of DNA or RNA. Conversely, if the hybrids that remain after washing under stringent hybridization conditions are destroyed or the amount of hybrids is significantly reduced, then the hybrids that were originally formed, i.e., under non-stringent hybridization conditions. It is shown that the hybrids formed were between different types of DNA or RNA.

The ability of HPV DNA or RNA to bind to an unknown sample of DNA or RNA under stringent hybridization conditions dictates a high degree of nucleotide sequence homology. On the other hand, the ability of HPV DNA or RNA to bind to an unknown sample of DNA or RNA only under non-stringent hybridization conditions indicates a low or intermediate degree of nucleotide sequence homology. The exact degree of nucleotide sequence homology can only be determined by directly sequencing the unknown DNA and comparing its R to the unknown sequence of HPV DNA.

HPV in an unknown sample of DNA or RNA, in particular an unknown sample of DNA or RNA derived from a genital lesion. In practicing the detection of DNA or RNA, it is advantageous to utilize a hybridization probe composition consisting of a mixture of hybridization probes. These hybridization probes consist of probes with sequences that represent all or most of the types suspected of being present in an unknown sample of DNA or RNA. Hybridization probe mixtures of DNA or RNA sequences representing HPV types 6, 11, 16, 18, 31, 33, 35, 43, 44 and C57 are the most found of these HPV types in genital lesions. It is particularly advantageous when deriving an unknown sample of DNA or RNA from a genital lesion, as it appears to be easy. Other known types of HPV are rarely or never found in genital lesions. For example, HPV types 1, 2 and 4 generally include other types of lesions, namely skin warts [Heilman, C.I. A. Et al., Found in the Journal of Virology (J. Virol.), 360: 395 (1980)], thus hybridizing probe mixtures of DNA or RNA sequences containing HPV types 1, 2 and 4 are DNA or RNA. An unknown sample of DNA or RNA may be useful when derived from a skin wart, but not when an unknown sample of DNA or RNA is derived from a genital lesion.

Examples of sequences of HPV types 6, 11, 16, 18, 31 and 33 that can be used in a mixture of hybridization probes include Gissman, L., Cancer Surv., 3. 161 (1984), Pfister, H. et al. Biochem. Pharmacol., 99: 111 (1983), Durst, M. et al. Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci. ) USA, 80:3812 (1983), Boshart, M.D. Et al., EMBO J. et al. 3: 1151 (1984), Lorincz, A. et al. T. Et al., Journal of Virology (J. Virol.), 58: 225 (1986) and Beaudenon, S. et al. , Nature, 321 and 246 (1986). In addition, examples of HPV types 1, 2 and 4 sequences that can be used in hybridization probe mixtures are well known in the art [see, Heilman, C.I. A. Et al., Journal of Virology (J. Virol.), 360: 395 (1980)]. Thus, disclosures herein regarding HPV 35, HPV 43, HPV 44 and HPV C57 and other HPV types relating to HPV types 6, 11, 16, 18, 31 and 33, such as HPV type, 2 Using the knowledge of those skilled in the art regarding and 4, a mixture of hybridization probes can be easily prepared.

In a mixture of hybridization probes, the particular percentage of DNA or RNA of each HPV type is not critical in the present invention. Generally, approximately equal molar amounts of DNA or RNA of each HPV type are used in the mixture.

As a means of detecting and determining the type of HPV, hybridization of nucleic acids is performed in solution as described above [Reference, Loggins, J. Mol. R. Et al., Cancer Research, 39: 545 (1979)] or on a solid support [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory, New York, Cold Spring Harbor (1982)] or on the spot [ See, Brigati, D.I. J. Et al., Virology (Virol.), 126:32 (1983) and Beckman, A. et al. M. Et al. (J. Med. Virol.), 16: 265 (1985)].

Hybridization on a solid support can be performed using a number of different procedures. One such procedure purifies all of the unknown DNA or RNA, immobilizes them on a solid support in single-stranded form, and then HPV 35 DNA or RNA or a fragment thereof, HPV 43 DNA or RNA or Includes hybridization with the fragment, HPV 44 DNA or RNA or fragment thereof, or HPVC57 DNA or RNA or fragment thereof.

Alternatively, the purified unknown DNA can be digested with one or more restriction endonucleases and the resulting DNA fragments in the sample can be electrophoretically separated. The DNA fragment is then transferred to a solid support and labeled HPV 35 DNA or RNA or fragment thereof, labeled HPV 43 DNA or RNA or fragment thereof, labeled HPV 44 DNA or RNA or fragment thereof, or labeled. It can hybridize with HPV C57 DNA or RNA or fragments thereof.

In-situ hybridization is performed on glass slides, and the final result of this procedure is viewed under a microscope. In this procedure, DNA or RNA is not purified from the cells and is left with all of the other cellular components.

HPV 35 RNA or fragments thereof, HPV 43 RNA or fragments thereof, HPV 44 RNA or fragments thereof, or HPV C57 RNA or fragments thereof are particularly more than purified DNA, especially DNA purified from genital lesions. Rather, when using crude extracts, especially those of crude genital lesions, it is preferred to use them as nucleic acid hybridization probes for HPV 35 DNA, HPV 43 DNA, and HPV 44 HPV C57 DNA, respectively. ..

Hybridization probes for detecting HPV 35 RNA, HPV 43 RNA, HPV 44 RNA or HPV C57 RNA, respectively, in HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA in an unknown sample of DNA. When used as, DNA-RNA hybrids formed after initial hybridization under stringent hybridization conditions are treated with pancreatic RNAase [approximately 20 mg / ml (pH 7.0) in 50 mmol of NaCl] at room temperature. Then, it is preferable to wash under stringent hybridization conditions.

HPV 35 DNA or a fragment thereof, HPV 43 DNA or a fragment thereof, HPV 44 DNA or a fragment thereof or HPV C57 DNA or a fragment thereof, or HPV 35 RNA or a fragment thereof, HPV 43 RNA or a fragment thereof, HPV 44 RNA or The fragment or HPV C57RNA or fragment thereof is particularly a radioactive marker, eg,³²P,¹⁴C,³H,¹²⁵I or³⁵When labeled with S, they are useful as nucleic acid hybridization probes for HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively.

HPV 35 DNA or a fragment thereof, HPV 43 DNA or a fragment thereof, HPV 44 DNA or a fragment thereof or HPV C57 DNA or a fragment thereof may be described, for example, in Rigby, P. et al. J. W. Et al., As described in the Journal of Molecular Biology (J. Mol. Biol.), 113: 237 (1977), by "nick-translation" by a well-known method, or by , For example, Deen, K. et al. C. Et al., As described in Analytical Biochemistry (Anal. Biochem.), 135: 456 (1983), can be radiolabeled by T4 DNA polymerase substitution synthesis.

HPV 35 RNA or fragments thereof, HPV 43 RNA or fragments thereof, HPV 44 RNA or fragments thereof or HPV C57 RNA or fragments thereof are described, for example, in Davanloo, P. et al. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 81: 2035 (1984), labeled with a radioactive marker by in vitro transcription. be able to. Since RNA polymerase can utilize labeled precursors, labeled RNA is synthesized by this method to generally synthesize HPV DNA or RNA, or in particular HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, Alternatively, HPV 35 RNA probe, HPV 43 RNA probe, HPV 44 RNA probe or HPV C57 RNA probe can be prepared for the detection of HPV C57 DNA or RNA, respectively. Labeled precursors that can be used to synthesize labeled RNA are radioactive markers, eg,³²P,¹⁴C,¹²⁵I or³⁵Includes precursors containing S.

HPV 35 DNA or a fragment thereof, HPV 43 DNA or a fragment thereof, HPV 44 DNA or a fragment thereof or HPV C57 DNA or a fragment thereof, or HPV 35 RNA or a fragment thereof, HPV 43 RNA or a fragment thereof, HPV 44 RNA or The fragment or HPV C57RNA or fragment thereof is also HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44, respectively, especially when labeled using non-radioactive markers such as biotin, enzyme or fluorescent group. DNA or RNA, or HPV C57 It is useful as a nucleic acid hybridization probe for DNA or RNA. Biotin acts as a hapten-like group and can bind to DNA or RNA, and binds avidin or streptavidin conjugation enzymes to biotin and then is washed to remove non-specifically bound enzymes. It can be detected by. With the addition of a suitable substrate for the enzyme, conversion of the substrate to a colored product can be detected [see, Leary, J. et al. J. Et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 80:4045 (1983)]. Examples of such enzymes include alkaline phosphatases and horseradish peroxidase. In addition, fluorescent molecules such as fluorescein and rhodamine can be chemically attached to avidin or streptavidin and used as non-radioactive markers.

Alternatively, the aforementioned enzyme or fluorescent molecule can be described, for example, in Lens, M. et al. , EMBO J., 6: 817 (1983), HPV35 DNA or a fragment thereof, HPV 43 DNA or a fragment thereof, HPV44 DNA or a fragment thereof or HPV C57 DNA or a fragment thereof, or HPV 35 RNA. Alternatively, it can be directly chemically conjugated to a fragment thereof, HPV 43 RNA or fragment thereof, HPV 44 RNA or fragment thereof or HPV C57 RNA or fragment thereof, and can be used as a hybridization probe in this manner.

HPV 35 DNA or a fragment thereof labeled in this way, HPV 43 DNA or a fragment thereof, HPV 44 DNA or a fragment thereof or HPV C57 DNA or a fragment thereof, or HPV 35 RNA or a fragment thereof, HPV 43 RNA or a fragment thereof. , HPV 44 RNA or fragments thereof or HPV C57 RNA or fragments thereof, as described above, studies of hybridization using unknown samples of DNA or RNA, especially DNA or RNA derived from genital lesions. It can be used in the sample to determine whether the sample generally contains HPV DNA or RNA, in particular HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA, respectively.

An unknown sample of DNA can be derived from other lesions, such as throat, mouth or skin lesions, in addition to being derived from genital lesions.

An unknown sample of DNA or RNA can be obtained, for example, by biopsy of an epithelial lesion, rubbing the neck, or swapping the neck to obtain exfoliated cells. In addition, unknown samples of DNA or RNA are described by Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory, New York, Cold Spring Harbor (1982) and Gissman, DNA or RNA from cells in which DNA from lesions was cloned by well-known means, such as described in L., Cancer Surv., 3: 161-181 (1984). Unknown sample can be obtained.

In the methods of the invention, the assay for cross-hybridization can be performed by assaying for the presence of radioactive or non-radioactive markers associated with said nucleic acid hybrids. The method for determining whether a particular marker is present depends on the marker used and is well known in the art.

HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA detection is an epidemic of cross-hybridation of unknown samples of DNA or RNA derived from genital lesions. In an embodiment of the invention, based on the distribution and comparison with that of HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA, respectively, an unknown sample of DNA or RNA is moderately stringent hybridization. Under conditions, ie, using the chromatography of hydroxyapatite to determine whether the two HPVs represent a common type of different isolates, or whether they represent different types of isolates, respectively, HPVs. It may show less than 50% cross-hybridation with 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA, and by definition here, still HPV 35 DNA or RNA, HPV 43 DNA or RNA, respectively. , HPV 44 It can be thought of as DNA or RNA, or HPV C57 DNA or RNA. Eventually, also in the sample, the intersection of each lesion constituting the epidemiological distribution to detect HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively. It is necessary to compare hybridization. This is because different types of HPV may show the same or similar epidemiological distribution. However, the same lesions that can constitute the epidemiological distribution cross-hybridize with both HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA, and an unknown sample of DNA or RNA derived from genital lesions, respectively. Samples of unknown DNA or RNA derived from genital lesions by demonstrating that are HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively. It is possible to clearly conclude.

As described in more detail below, HPV 35 DNA or RNA has been found to be present in approximately 1% to 4% of cervical lesions ranging from mild dysplasia to invasive cancer. Thus, HPV 35 DNA or RNA is rather evenly distributed in various grades of cervical lesions.

HPV 43 DNA or RNA was found to be present in approximately 1% to 4% of amphoteric cervical lesions (eg, mild dysplasia), but not in invasive cancers. .. Thus, HPV 43 DNA or RNA appears to be present only in lower grade cervical lesions.

HPV 44 DNA or RNA was found to be present in approximately 1% to 4% of amphoteric cervical lesions, but was not found in invasive cancers. Thus, HPV 44 DNA or RNA appears to be present only in lower grade cervical lesions.

HPV C57 DNA or RNA was found to be present in approximately 1% to 4% of amphoteric cervical lesions and was found in 4% of invasive cancers. Thus, HPV C57 DNA or RNA appears to be present in lower grade cervical lesions and cancers. On the other hand, other HPV types, such as HPV types 6, 11, 16, 18 and 31, show different distributions in different grades of cervical lesions.

Thus, the detection of HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA is cross-hybridation of an unknown sample of DNA or RNA derived from genital lesions. In an embodiment of the invention, an unknown sample of DNA or RNA derived from genital lesions, based on the epidemiological distribution of HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA, respectively. Will cross-hybridize with cervical lesions. If such an epidemiological distribution of cross-hybridation is found in an unknown sample of DNA or RNA derived from genital lesions, the unknown sample of this DNA or RNA will be HPV 35 DNA or RNA, HPV, respectively. 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 It may be DNA or RNA. Unknown DNA or RNA derived from genital lesions by demonstrating that the same lesions that make up the epidemiological distribution also cross-hybride with an unknown sample of DNA or RNA derived from genital lesions. It can be concluded that the samples are HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, respectively.

HPV 35 DNA or HPV 35 RNA fragment, HPV 43 DNA or HPV 43 RNA fragment, HPV 44 DNA or HPV 44 RNA fragment, or HPV C57 DNA or HPV C57 RNA that can be used as a hybridization probe in the present invention. The specific size of the fragments of is not critical. The size of a fragment of HPV 35 DNA or HPV 35 RNA, a fragment of HPV 43 DNA or HPV 43 RNA, a fragment of HPV 44 DNA or HPV 44 RNA, or a fragment of HPV C57 DNA or HPV C57 RNA is single-stranded or double-stranded. Depending on whether or not the probe is used, it can be about 15 to about 8000 bases or base pairs, preferably about 300 to about 800 bases or base pairs. When hybridization is performed in situ, HPV 35 DNA or HPV 35 RNA fragment, HPV 43 DNA or HPV 43 RNA fragment, HPV 44 DNA or HPV 44 RNA fragment, or HPVC 57 DNA or HPV C57. The size of the RNA fragment is preferably smaller than about 500 bases or base pairs. This is because fragments of this size hybridize in-situ more efficiently than HPV DNA or HPV RNA larger than about 1000 bases or base pairs. When using double-stranded DNA or RNA, the DNA or RNA must be denatured prior to hybridization.

HPV 35 DNA Fragment, HPV 43 DNA Fragment, HPV 44 DNA Fragment or HPV C57 DNA Fragment are HPV 35 clones 2A and 2B, HPV 43 clones 2A and 2B, HPV 44 clone 2, respectively. Alternatively, by digestion of the limiting endonuclease of clone 2A or 2B of HPV C57, or by synthetically producing such such using a commercially available synthesizer, or by the well-known means [Sanger (Sanger). Sanger), S.A. , Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci.) USA, 74: 5363 (1977)] HPV 35 DNA sequence, HPV 43 DNA sequence, HPV 44 It can be obtained by a well-known chemical method using a sequence of DNA or a sequence of HPV C57 DNA.

When detecting HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, the HPV 35 genome, HPV 43 genome, HPV 44 genome, or HPV C57 genome, respectively. It is preferred to use substantially all as a hybridization probe.

The present invention will be further described with reference to the following examples. These examples are not intended to limit the scope of the invention. Unless otherwise noted, all parts, percentages, ratios, etc. depend on weight.

【００８２】

[Example]

Example 1 (A) Cloning of HPV 35 DNA The starting material used was Washington, D.C., which consisted of several milligrams of tissue. C. It was a biopsy of cervical adenocarcinoma obtained from. The total DNA is Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982). More specifically, the tissue is subdivided and then contains 1.0 ml of 50 mmol Tris-HCl, pH 8.0 [0.6% (w / v) sodium dodecyl sulfate and 50 μg / ml Proitenase K]. Digested in 37 ° C. overnight. The resulting digest was extracted twice with 1.0 ml phenol: chloroform (1: 1 (v / v)). The DNA was then precipitated from the aqueous phase by the addition of 2 volumes of 90% (v / v) ethanol. The precipitated DNA was redissolved in 10 mmol Tris, 1.0 mmol EDTA buffer, pH 8.0 (hereinafter "TE buffer") at a concentration of about 1.0 mg / ml.

The DNA was completely digested with PstI and electrophoresed on a 1% (v / v) agarose gel, and the DNA was transferred to Southern, E. coli. M. , Journal of Molecular Biology (J. Mol. Biol. ), 98: 503 (1975), transferred to a nitrocellulose filter. The filter was then probed with DNA from HPV types 6, 11, 16, 18 and 31 under non-stringent hybridization conditions (Tm-35 ° C.) and stringent hybridization conditions (Tm-10 ° C.). Hybridization was performed at 43 ° C. with 1.0 mol of NaCl, 50 mmol of sodium phosphate buffer (pH 7.4), 1.0 mmol of EDTA, 2% (w / v) of sodium dodecyl sulfate, 0. It was performed overnight in 1% (w / v) gelatin, 50 μg / ml tRNA and 30% (w / v) formamide. Wash for 30 minutes at 55 ° C. 1.2 x SSC (1 x SSC is 0.15 mol NaCl + 0.015 mol sodium citrate), 10 mmol sodium phosphate (pH 7.4), 1.0 It was performed in mmol EDTA and 0.5% (w / v) sodium dodecyl sulfate. Hybridization was achieved with HPV types 6, 11, 16, 18 and 31 under non-stringent conditions, but not under stringent hybridization conditions.

The obtained purified DNA and λ L47 were digested with a BamHI-restricted endonuclease. This produced fragments of 3.75 kb and 4.1 kb, the sum of which, 7.85 kb, was typical for the papillomavirus genome. Each of these fragments was cloned into a single BamHI site at λ L47. More specifically, 2.0 μg of the obtained purified DNA and 2.0 μg of λ L47 DNA were cleaved with 10 units of BamHI in a total volume of 50 μl of TE buffer at 37 ° C. for 1 hour. The resulting reaction mixture was then diluted with 400 μl TE buffer and phenol extracted with equal volumes of phenol: chloroform as described above. The aqueous phase was then extracted with chloroform: isoamyl alcohol (24: 1 (v / v)), and the DNA from the aqueous phase was precipitated with 80% (v / v) ethanol and dried. The dried DNA was then added to 10 μl of 66 mmol Tris-HCl, 6.6 mmol MgCl.₂Suspended in 1 × ligase buffer consisting of 10 mmol DTT and 1.0 mmol ATP and incubated at 42 ° C. for 2 hours to anneal the λ arm. Next, 0.5 μl of T4 DNA ligase, about 1 unit, and 0.5 μl of 10 mmol of ATP, pH 7.0, were added to the reaction solution and binding was allowed to proceed overnight at 12 ° C.

Next, the binding products are packaged to form infectious phage, and E.I. Used to infect coli NM538. More specifically, E. coli growing on an agarose plate (hereinafter "TN medium") consisting of 10 g / l tryptone and 5.0 g / l NaCl. Single colonies of coli NM538 were selected and grown in 20 ml TN medium on a shaking platform (250 rpm) at 37 ° C. overnight in the early stationary phase. The cell culture was then diluted 4-fold in TN medium and grown for 3 hours. The cells are then harvested by centrifugation at 5,000 rpm for 5 minutes in a Sobalall HB-4 rotor, and the resulting cell precipitate is 10 mmol MgSO.₄It was resuspended in the original volume of 0.25 and stored at 4 ° C.

Packaged infectious phage were prepared with a commercially available BRL Lambda in vitro packaging system [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)].

0.5 mol Tris-HCl (pH 8.0), 0.1 mol NaCl, 0.01 mol MgSO.₄And 1.5 × 10 in phage storage buffer (Difco) consisting of 0.01% (w / v) gelatin⁴100 μl of a suitable dilution of the packaged phage, appropriately diluted in a plaque / 9 cm diameter plate, prepared as described above in a 10-15 ml test tube. It was added to 100 μl of coli NM538, mixed gently and incubated at room temperature for 15 minutes. The cell-phage solution was then prepared with a trypticase soy broth agar plate containing 10 g of trypticase soy broth, 5.0 g of NaCl and 15 g of agar per liter (which was prepared at least 1 day and preheated to 37 ° C. ) Plated on. Then 10 mmol MgSO₄Overlay of agarose consisting of 0.5% agarose (ultra-pure, electrophoretic grade) dissolved in it (heated in a microwave oven until the solution boils, then cooled to 45 ° C. before use. 3-5 ml of (is) was placed on the plated cells-phage. After the agarose had solidified, the flat plate was transferred to a well-circulated ventilator at 37 ° C., where cracks were formed in the lid of the flat plate for 30 minutes, the lid was closed, and the flat plate was inverted. After 8-12 hours, plaques began to appear.

The infection resulted in total lysis of bacteria on a flat plate. Recombinant phage with HPV DNA are described by Benton, W. et al. D. Et al., Science, 196: 180 (1977), "plaque". It was localized by performing "lifts)". More specifically, a flat plate of totally lysed bacteria was placed at 4 ° C. for 1 hour to solidify the agarose. An appropriately sized piece of nitrocellulose filter was then placed in contact with the center of the plate by bending in its center and by moving the center point towards the edge. Four asymmetric pores were then drilled through a nitrocellulose filter and agar with a small gauge needle, and the location of the pores was marked on the bottom of the plate with a permanent marker. This allowed the nitrocellulose filter and other regions containing a positive signal to be associated with their corresponding positions on the plate. After 10 minutes, the nitrocellulose filter is removed and the nitrocellulose filter is placed in a dish containing 200 ml 0.5 mol NaOH, 2.0 mol NaCl for 1 minute with the plaques facing upwards. By doing so, the DNA was denatured. The nitrocellulose filter was then neutralized by immersing in 500 ml of 0.5 mol Tris-HCl, 2.0 mol NaCl, pH 7.5 for 5 minutes. The filter was then rinsed in 6 × SSC consisting of 0.9 mol NaCl, 0.09 mol sodium citrate for 1 minute, dried on Watman 3MM paper and then baked under vacuum at 80 ° C. for 30 minutes.

Then, as a probe, by "nick translation"³²Non-stringent hybridization using P-labeled HPV 16 DNA was performed on DNA separated from lifted plaques [see, Rigby, P. et al. J. W. Et al., Journal of Molecular Biology (J. Mol. Biol.), 113: 237 (1977) and Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982)], followed by cleaning and autoradiography. More specifically, the hybridization was carried out with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid (hereinafter "TES"). It was carried out at 41 ° C. in a solution consisting of 10 × Denhardt solution, 0.1 mmol EDTA and 10 mmol sodium phosphate (pH 7.4). Non-stringent hybridization was then carried out in 10 mmol sodium phosphate (pH 7.4), 1.1 x SSC in 0.1 mmol EDTA (0.165 mol NaCl and 0.0165 mol sodium citrate). Was performed at 52 ° C. using (consisting of).

By associating with the site of radioactive exposure, a region of the phage-containing plate containing the DNA hybridized to HPV 16 DNA was excised, and E. coli as described above. Used to reinfect coli NM538. Localization of plaques on phage containing HPV DNA was achieved by repeating the above procedure. Screened 2x10⁵Seven plaques were identified and three types of clones were found. Two clones showed a 3.75 kb insert, two clones showed a 4.1 kb insert, and two clones showed a 4.3 kb insert. All similarly sized clones had the same restricted map. The 3.75 kb and 4.1 kb clones did not cross-hybridize. However, the 4.3 kb clone was found to be homologous to the 3.75 kb clone and also to the DNA of the human genome. As suggested, these clones contained a conjugation fragment between the integrated copy of human DNA and HPV 35 DNA and thus were not further analyzed. A clone containing a 3.75 kb fragment was labeled HPV 35 clone 1A, and a clone containing a 4.1 kb fragment was labeled HPV 35 clone 1B.

The HPV DNA of HPV 35 clones 1A and 1B was then digested with BamHI and subcloned in a single BamHI site of pBR322. The resulting recombinant DNA was labeled HPV 35 clones 2A and 2B. HPV 35 clones 2A and 2B are included in the American Type Culture Collection, respectively, at ATCC No. 40330 and ATCC No. It was commissioned at 40331.

(B) Characterizing HPV 35 DNA 1. Hybridization studies Hybridization studies were performed on HPV 35 clones 2A and 2B DNA to show that HPV 35 clones 2A and 2B are new types of HPV. Proven.

More specifically, it was prepared from HPV 35 clones 2A and 2B.³²P "nick-translated" DNA was hybridized to 5.0 ng of DNA from HPV types 1-34 by Southern blotting under stringent conditions. DNA from HPV types 1-34 includes Dr. Gerard Orth of the Institute Pasteur (Paris, France), the assignor of the HPV type label, and the Papilloma Reference. Ethel-Michelle at the Center (Pappilloma Reference Center) (Heidelberg, West Germany) Obtained from Dr. deVilliers) in pre-immobilized form on a nitrocellulose filter. More specifically, hybridization was performed at 41 ° C. with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-Tris TES, 10 × Denhardt solution, 0.5 mmol of EDTA and 20. It was carried out in a solution consisting of mmol sodium phosphate (pH 7.4). A stringent wash was then performed at 65 ° C. with 10 mmol sodium phosphate (pH 7.4), 0.03 x SSC in 0.1 mmol EDTA (0.0045 mol NaCl and 0.00045 mol). It consisted of sodium citrate).

Significant homology was detected between the recombinant DNA of HPV 35 clones 2A and 2B and HPV types 6, 11, 16, 18 and 31 under non-stringent hybridization conditions, but stringent. No homology was observed with any of HPV types 1-34 under hybridization conditions, thus demonstrating that HPV 35 clones 2A and 2B represent the new HPV type.

2. Restricted endonuclease map Figure 1 shows a restricted endonuclease map for HPV 35. The following restriction endonuclease did not cleave HPV 35 DNA: SphI, XabI, NcoI and HPaI.

3. Genome Composition The following hybridization studies were performed to demonstrate that the genome of HPV 35 has the same or similar open reading frame to HPV 6. DNA purified from HPV 35 clones 2A and 2B was used as a probe under non-stringent and stringent conditions as described above for HPV 6 DNA.³²P "Nick-translated" fragments were used for Southern blotting. More specifically, fragments of the BamHI linearized HPV 6b clone were generated by EcoRI and PstI and then gel purified.³²Nick-translated with P and used to probe Southern blots containing: (a) PstI restriction digest of a purified BamHI fragment of HPV 35 clone 2A DNA or (b) HPV 35 clone 2B DNA PvuII-PstI Restriction Digest of Purified BamHI Fragments. As evidenced by the results shown in FIG. 2, the DNA of HPV 35 clones 2A and 2B has the same genomic composition as HPV 6.

4. Epidemiological distribution Hybridization probes prepared from HPV 35 clones 2A and 2B efficiently hybridize only to HPV 35 DNA under stringent conditions, and these hybridization probes are HPV 35. To detect genital lesions containing genital lesions and to distinguish such genital lesions from genital lesions containing DNA of other HPV types such as 6, 11, 16, 18, 31 or 33. To prove that it can be used in Washington, D.C. C. DNA from cervical biopsies and cervical swab collections containing exfoliated cells from the metropolitan area (including Mariyland and Virginia) and Michigan and surrounding states, under stringent conditions and Specific HPV using probes specific for different HPV types, including probes specific for HPV35, by hybridization of nucleic acids under non-stringent conditions. The presence of DNA was analyzed. The biopsy was bisected, half of this sample was processed for a normal light microscope, and the other half was frozen and stored at 20 ° C. for molecular analysis. Tissues undergoing Southern blot hybridization were partitioned on cryopreservation devices to obtain material for DNA extraction. Almost all 1/18 compartments were colored with hematoxylin and eosin coloring and microscopically examined to confirm that the sample of this tissue was comparable to some of the samples analyzed by light microscopy. Exfoliated cervical cells were analyzed for paired samples by standard cytological methods, such as pap smear, the other of which was used for DNA analysis.

High molecular weight DNA was prepared from the sample as described above. 1-10 μg of purified cellular DNA was digested with PstI or BamHI, and the digested sample was electrophoresed on a 1.0% (w / v) agarose gel and transferred to a nitrocellulose filter. Hybridization was then nick-translated from the types described above under stringent conditions as described above.³²It was performed using P-labeled HPV DNA. (For HPV 35 DNA, a mixture of HPV DNA from HPV 35 clones 2A and 2B was used.) Note: Since HPV DNA grows in pBR322 or related vectors, pBR322 and related vector-like sequences in tissue samples. To minimize the possibility of reaction with, all probes were electropurified to remove most of the vector sequences involved. Additional hybridization was also performed in the presence of labeled pBR322 and related vectors to reveal potential false positive material due to plasmid-like sequences in tissue samples. The results are shown in Table 1 below. The results in Table 1 are shown graphically in FIG.

【００９９】

[Table 1] <img file="000002.tif" id="000002" he="195" wi="135" img-format="tif" img-content="drawing" />

As evidenced by Tables 1 and 3, cervical biopsies containing HPV 35 are not only by reference to the degree of cross-hybridization in solution and by subsequent chromatography of hydroxyapatite. And also by the ability of the HPV 35 DNA probe to specifically detect and identify a well-defined population of genital lesions, i.e. those containing HPV35 DNA as compared to those containing other types of HPV. , Which can be distinguished from biopsies containing other types of HPV, such as 6, 11, 16, 18, 31 and 33. In addition, as the results in Tables 1 and 3 show, the epidemiological distribution of HPV 35 DNA between biopsies in the neck is distinguished from that found for some other HPV types.

Example 2 (A) Cloning of HPV 43 DNA The starting material used was a biopsy of vulvar hyperkeratosis from Michigan, consisting of several milligrams of tissue. The total DNA is Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982). More specifically, the tissue is subdivided and then contains 1.0 ml of 50 mmol Tris-HCl, pH 8.0 [0.6% (w / v) sodium dodecyl sulfate and 50 μg / ml Proitenase K]. Digested in 37 ° C. overnight. The resulting digest was extracted twice with 1.0 ml phenol: chloroform (1: 1 (v / v)). The DNA was then precipitated from the aqueous phase by the addition of 2 volumes of 90% (v / v) ethanol. The precipitated DNA was redissolved in 10 mmol Tris, 1.0 mmol EDTA buffer, pH 8.0 (hereinafter "TE buffer") at a concentration of about 1.0 mg / ml.

The DNA was completely digested with PstI and electrophoresed on a 1% (v / v) agarose gel, and the DNA was transferred to Southern, E. et al. M. , Journal of Molecular Biology (J. Mol. Biol. ), 98: 503 (1975), transferred to a nitrocellulose filter. The filter was then probed with DNA from HPV types 6, 11, 16, 18 and 31 under non-stringent hybridization conditions (Tm-35 ° C.) and stringent hybridization conditions (Tm-10 ° C.). Hybridization was performed at 43 ° C. with 1.0 mol of NaCl, 50 mmol of sodium phosphate buffer (pH 7.4), 1.0 mmol of EDTA, 2% (w / v) of sodium dodecyl sulfate, 0. It was performed overnight in 1% (w / v) gelatin, 50 μg / ml tRNA and 30% (w / v) formamide. Wash for 30 minutes at 55 ° C. 1.2 x SSC (1 x SSC is 0.15 mol NaCl + 0.015 mol sodium citrate), 10 mmol sodium phosphate (pH 7.4), 1.0 It was performed in mmol EDTA and 0.5% (w / v) sodium dodecyl sulfate. Hybridization was achieved with HPV types 6, 11, 16, 18 and 31 under non-stringent conditions, but not under stringent hybridization conditions.

The resulting purified DNA and λ L47 were digested with BamHI-restricted endonuclease (which produced a 6.3 kb fragment) or HindIII (which produced a 2.85 kb fragment). The total, or 9.15 kb, is larger than the papillomavirus genome. Mapping showed that the 6.3 kb and 2.85 kb fragments overlap for 1.55 kb of their length. The amount of non-overlapping HPV sequences represented by the 6.3 kb and 2.85 kb fragments is 7.6 kb, or approximately 96% of the typical size of the HPV genome. More specifically, 2.0 μg of the obtained purified DNA and 2.0 μg of λ L47 DNA were cleaved with 10 units of BamHI in a total volume of 50 μl of TE buffer at 37 ° C. for 1 hour. The resulting reaction mixture is then subjected to 400 μl TE. It was diluted with buffer and phenol extracted with equal volume of phenol: chloroform as described above. The aqueous phase was then extracted with chloroform: isoamyl alcohol (24: 1 (v / v)), and the DNA from the aqueous phase was precipitated with 80% (v / v) ethanol and dried. The dried DNA was then added to 10 μl of 66 mmol Tris-HCl and 6.6 mmol of MgCl.₂Suspended in 1 × ligase buffer consisting of 10 mmol DTT and 1.0 mmol ATP and incubated at 42 ° C. for 2 hours to anneal the λ arm. Next, 0.5 μl of T4 DNA ligase, about 1 unit, and 0.5 μl of 10 mmol of ATP, pH 7.0, were added to each reaction solution and binding was allowed to proceed overnight at 12 ° C. ..

Next, the binding products were packaged to form infectious phage, and E.I. Used to infect coli NM538. More specifically, E. coli growing on an agarose plate (hereinafter "TN medium") consisting of 10 g / l tryptone and 5.0 g / l NaCl. Single colonies of coli NM538 were selected and grown in 20 ml TN medium on a shaking platform (250 rpm) at 37 ° C. overnight in the early stationary phase. The cell culture was then diluted 4-fold in TN medium and grown for 3 hours. The cells are then harvested by centrifugation at 5,000 rpm for 5 minutes in a Sobalall HB-4 rotor, and the resulting cell precipitate is 10 mmol MgSO.₄It was resuspended in the original volume of 0.25 and stored at 4 ° C.

Packaged infectious phage were prepared with a commercially available BRL Lambda in vitro packaging system [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)].

0.5 mol Tris-HCl (pH 8.0), 0.1 mol NaCl, 0.01 mol MgSO.₄And 1.5 × 10 in phage storage buffer (Difco) consisting of 0.01% (w / v) gelatin⁴100 μl of a suitable dilution of the packaged phage, appropriately diluted in a plaque / 9 cm diameter plate, prepared as described above in a 10-15 ml test tube. It was added to 100 μl of coli NM538, mixed gently and incubated at room temperature for 15 minutes. The cell-phage solution was then prepared with a trypticase soy broth agar plate containing 10 g of trypticase soy broth, 5.0 g of NaCl and 15 g of agar per liter (which was prepared at least 1 day and preheated to 37 ° C. ) Plated on. Then 10 mmol MgSO₄Overlay of agarose consisting of 0.5% agarose (ultra-pure, electrophoretic grade) dissolved in it (heated in a microwave oven until the solution boils, then cooled to 45 ° C. before use. 3-5 ml of (is) was placed on the plated cells-phage. After the agarose had solidified, the flat plate was transferred to a well-circulated ventilator at 37 ° C., where cracks were formed in the lid of the flat plate for 30 minutes, the lid was closed, and the flat plate was inverted. After 8-12 hours, plaques began to appear.

[0107] The infection resulted in total lysis of bacteria on a flat plate. HPV Recombinant phage with DNA are described by Benton, W. et al. D. Et al., As described in Science, 196: 180 (1977), localized by performing "plaquelifts". More specifically, a flat plate of totally lysed bacteria was placed at 4 ° C. for 1 hour to solidify the agarose. An appropriately sized piece of nitrocellulose filter was then placed in contact with the center of the plate by bending in its center and by moving the center point towards the edge. Four asymmetric pores were then drilled through a nitrocellulose filter and agar with a small gauge needle, and the location of the pores was marked on the bottom of the plate with a permanent marker. This allowed the nitrocellulose filter and other regions containing a positive signal to be associated with their corresponding positions on the plate. After 10 minutes, the nitrocellulose filter is removed and the nitrocellulose filter is placed in a dish containing 200 ml 0.5 mol NaOH, 2.0 mol NaCl for 1 minute with the plaques facing upwards. By doing so, the DNA was denatured. The nitrocellulose filter was then neutralized by immersing in 500 ml of 0.5 mol Tris-HCl, 2.0 mol NaCl, pH 7.5 for 5 minutes. The filter was then rinsed in 6 × SSC consisting of 0.9 mol NaCl, 0.09 mol sodium citrate for 1 minute, dried on Watman 3MM paper and then baked under vacuum at 80 ° C. for 30 minutes.

[0108] Then, as a probe, by "nick translation"³²Non-stringent hybridization using P-labeled HPV 16 DNA was performed on DNA separated from lifted plaques [see, Rigby, P. et al. J. W. Et al., Journal of Molecular Biology (J. Mol. Biol.), 113: 237 (1977) and Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982)], followed by cleaning and autoradiography. More specifically, the hybridization was carried out with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid (hereinafter "TES"). It was carried out at 41 ° C. in a solution consisting of 10 × Denhardt solution, 0.1 mmol EDTA and 10 mmol sodium phosphate (pH 7.4). Non-stringent hybridization was then carried out in 10 mmol sodium phosphate (pH 7.4), 1.1 x SSC in 0.1 mmol EDTA (0.165 mol NaCl and 0.0165 mol sodium citrate). Was performed at 52 ° C. using (consisting of).

By associating with the site of radioactive exposure, a region of the phage-containing plate containing the DNA hybridized to HPV 16 DNA was excised, and E. coli as described above. Used to reinfect coli NM538. Localization of plaques on phage containing HPV DNA was achieved by repeating the above procedure. 1.65 × 10 screened from cloning using BamHI digested DNA⁵One plaque was identified from among the plaques. The cloned fragment showed a size of 6.3 kb and was labeled HPV 43 clone 1A. 9x10 screened from cloning using HindIII digested DNA⁴One plaque was identified from among the plaques. The cloned fragment showed a size of 2.85 kb and was labeled HPV 43 clone 1B.

HPV DNA of HPV 43 clones 1A and 1B was then digested with BamHI or HindIII, respectively, and subcloned into a single BamHI or HindIII site of pBR322. The resulting recombinant DNA was labeled HPV 43 clones 2A and 2B. HPV 43 clones 2A and 2B are included in the American Type Culture Collection, respectively, at ATCC No. 40338 and ATCC No. It was commissioned at 40339.

(B) HPV 43 DNA characterization 1. Hybridization studies Hybridization studies were performed on HPV 43 clones 2A and 2B DNA to show that HPV 43 clones 2A and 2B are new types of HPV. Proven.

More specifically, it was prepared from HPV 43 clones 2A and 2B.³²P "nick-translated" DNA was hybridized to 5.0 ng of DNA from HPV types 1-42 by Southern blotting under stringent conditions. DNA from HPV types 1-42 is available from Dr. Gerard Orth of the Institute Pasteur (Paris, France), the transferor of the HPV type label, the Papilloma Reference Center. Pre-fixed on a nitrocellulose filter from Dr. Ethel-Michelle de Villiers of (Pappilloma Reference Center) (Heidelberg, West Germany) and from Life Technologies, Inc. Obtained in a modified form. More specifically, hybridization was performed at 41 ° C. with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris. It was carried out in a solution consisting of TES, 10 × Denhardt solution, 0.5 mmol of EDTA and 20 mmol of sodium phosphate (pH 7.4). A stringent wash was then performed at 65 ° C. with 10 mmol sodium phosphate (pH 7.4), 0.03 x SSC in 0.1 mmol EDTA (0.0045 mol NaCl and 0.00045 mol). It consisted of sodium citrate).

Significant homology was detected under non-stringent hybridization conditions between the recombinant DNA of HPV 43 clones 2A and 2B and other types of HPV, but under stringent hybridization conditions HPV. No homology was observed using any of types 1-42 of, thus demonstrating that HPV 43 clones 2A and 2B represent the new type of HPV.

2. Restricted endonuclease map Figure 4 shows a restricted endonuclease map for HPV 43 clones 2A and 2B. The following restriction endonuclease did not cleave HPV 43 DNA: EcoRI and SphI.

3. Genome Composition To demonstrate that the genome of HPV 43 has the same or similar open reading frame to HPV 6, the following hybridization studies were performed. DNA purified from HPV 43 clones 2A and 2B was used as a probe under non-stringent and stringent conditions as described above for HPV 6 DNA.³²P "Nick-translated" fragments were used for Southern blotting. More specifically, fragments of the BamHI linearized HPV 6b clone were generated by EcoRI and PstI and then gel purified.³²Nick-translated with P and used to probe Southern blots containing: (a) PstI restriction digest of a purified BamHI fragment of HPV 43 clone 2A DNA or purified HPV 43 clone 2B DNA PstI restriction digest of HindIII fragment. As the results shown in FIG. 5 substantiate, the DNA of HPV 43 clones 2A and 2B has the same genomic composition as HPV 6.

4. Epidemiological distribution Hybridization probes prepared from HPV 43 clones 2A and 2B efficiently hybridize only to HPV 43 DNA under stringent conditions, and these hybridization probes are HPV 43. To detect genital lesions containing genital lesions and to distinguish such genital lesions from genital lesions containing DNA of other HPV types such as 6, 11, 16, 18, 31 or 33. To prove that it can be used in Washington, D.C. C. DNA from cervical biopsies and cervical swab collections containing exfoliated cells from the metropolitan area (including Mariyland and Virginia) and Michigan and surrounding states, under stringent conditions and Specific HPVs using probes specific for different HPV types, including probes specific for HPV43 by hybridization of nucleic acids under non-stringent conditions. The presence of DNA was analyzed. The biopsy was bisected, half of this sample was processed for a normal light microscope, and the other half was frozen and stored at 20 ° C. for molecular analysis. Tissues undergoing Southern blot hybridization were partitioned on cryopreservation devices to obtain material for DNA extraction. Almost all 1/18 compartments were colored with hematoxylin and eosin coloring and microscopically examined to confirm that the sample of this tissue was comparable to some of the samples analyzed by light microscopy. Exfoliated cervical cells were analyzed for paired samples by standard cytological methods, such as pap smear, the other of which was used for DNA analysis.

High molecular weight DNA was prepared from the sample as described above. 1-10 μg of purified cellular DNA was digested with PstI or BamHI, and the digested sample was electrophoresed on a 1.0% (w / v) agarose gel and transferred to a nitrocellulose filter. Hybridization was then nick-translated from the types described above under stringent conditions as described above.³²It was performed using P-labeled HPV DNA. (For HPV 43 DNA, a mixture of HPV DNA from HPV 43 clones 2A and 2B was used.) Note: HPV DNA grows in pT713, pBR322 or related vectors, so pT713, pBR322 and in tissue samples. To minimize the possibility of reaction with the vector-like sequences involved, all probes were electropurified to remove most of the vector sequences involved. Additional hybridization was also performed in the presence of labeled pBR322 and related vectors to reveal potential false positive material due to plasmid-like sequences in tissue samples. The results are shown in Table 2 below. The results in Table 2 are shown graphically in FIG.

【０１１８】

[Table 2] <img file="000003.tif" id="000003" he="195" wi="135" img-format="tif" img-content="drawing" />

As evidenced by Tables 2 and 6, cervical biopsies containing HPV 43 were obtained not only by reference to the degree of cross-hybridization in solution and by subsequent chromatography of hydroxyapatite. And also by the ability of the HPV 43 DNA probe to specifically detect and identify a well-defined population of genital lesions, i.e. those containing HPV 43 DNA as compared to those containing other types of HPV. , Which can be distinguished from biopsies containing other types of HPV, such as 6, 11, 16, 18, 31 and 33. In addition, as the results in Table 2 and FIG. 6 show, the epidemiological distribution of HPV 43 DNA between biopsies in the neck is distinguished from that found for some other HPV types.

Example 3 (A) Cloning of HPV 44 DNA The starting material used was a biopsy of vulvar condyloma from Michigan, consisting of several milligrams of tissue. The total DNA is Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982). More specifically, the tissue is subdivided and then contains 1.0 ml of 50 mmol Tris-HCl, pH 8.0 [0.6% (w / v) sodium dodecyl sulfate and 50 μg / ml Proitenase K]. Digested in 37 ° C. overnight. The resulting digest was extracted twice with 1.0 ml phenol: chloroform (1: 1 (v / v)). The DNA was then precipitated from the aqueous phase by the addition of 2 volumes of 90% (v / v) ethanol. The precipitated DNA was redissolved in 10 mmol Tris, 1.0 mmol EDTA buffer, pH 8.0 (hereinafter "TE buffer") at a concentration of about 1.0 mg / ml.

The DNA was completely digested with PstI and electrophoresed on a 1% (v / v) agarose gel, and the DNA was transferred to Southern, E. et al. M. , Journal of Molecular Biology (J. Mol. Biol. ), 98: 503 (1975), transferred to a nitrocellulose filter. The filter was then probed with DNA from HPV types 6, 11, 16, 18 and 31 under non-stringent hybridization conditions (Tm-35 ° C.) and stringent hybridization conditions (Tm-10 ° C.). Hybridization was performed at 43 ° C. with 1.0 mol of NaCl, 50 mmol of sodium phosphate buffer (pH 7.4), 1.0 mmol of EDTA, 2% (w / v) of sodium dodecyl sulfate, 0. It was performed overnight in 1% (w / v) gelatin, 50 μg / ml tRNA and 30% (w / v) formamide. Wash for 30 minutes at 55 ° C. 1.2 x SSC (1 x SSC is 0.15 mol NaCl + 0.015 mol sodium citrate), 10 mmol sodium phosphate (pH 7.4), 1.0 It was performed in mmol EDTA and 0.5% (w / v) sodium dodecyl sulfate. Hybridization was achieved with HPV types 6, 11, 16, 18 and 31 under non-stringent conditions, but not under stringent hybridization conditions.

The resulting purified DNA and λ L47 were digested with a BamHI-restricted endonuclease, which produced a 7.8 kb fragment. This is typical of the papillomavirus genome. This fragment was cloned into a single BamHI of λ L47. More specifically, 2.0 μg of the obtained purified DNA and 2.0 μg of λ L47 DNA were cleaved with 10 units of BamHI in a total volume of 50 μl of TE buffer at 37 ° C. for 1 hour. The resulting reaction mixture was then diluted with 400 μl TE buffer and phenol extracted with equal volumes of phenol: chloroform as described above. The aqueous phase was then extracted with chloroform: isoamyl alcohol (24: 1 (v / v)), and the DNA from the aqueous phase was precipitated with 80% (v / v) ethanol and dried. The dried DNA was then added to 10 μl of 66 mmol Tris-HCl and 6.6 mmol of MgCl.₂Suspended in 1 × ligase buffer consisting of 10 mmol DTT and 1.0 mmol ATP and incubated at 42 ° C. for 2 hours to anneal the λ arm. Next, 0.5 μl of T4 DNA ligase, about 1 unit, and 0.5 μl of 10 mmol of ATP, pH 7.0, were added to each reaction solution and binding was allowed to proceed overnight at 12 ° C. ..

Next, the binding products were packaged to form infectious phage, and E.I. Used to infect coli NM538. More specifically, E. coli growing on an agarose plate (hereinafter "TN medium") consisting of 10 g / l tryptone and 5.0 g / l NaCl. Single colonies of coli NM538 were selected and grown in 20 ml TN medium on a shaking platform (250 rpm) at 37 ° C. overnight in the early stationary phase. The cell culture was then diluted 4-fold in TN medium and grown for 3 hours. The cells are then harvested by centrifugation at 5,000 rpm for 5 minutes in a Sobalall HB-4 rotor, and the resulting cell precipitate is 10 mmol MgSO.₄It was resuspended in the original volume of 0.25 and stored at 4 ° C.

Packaged infectious phage were prepared with a commercially available BRL Lambda in vitro packaging system [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)].

0.5 mol Tris-HCl (pH 8.0), 0.1 mol NaCl, 0.01 mol MgSO.₄And 1.5 × 10 in phage storage buffer (Difco) consisting of 0.01% (w / v) gelatin⁴100 μl of a suitable dilution of the packaged phage, appropriately diluted in a plaque / 9 cm diameter plate, prepared as described above in a 10-15 ml test tube. It was added to 100 μl of coli NM538, mixed gently and incubated at room temperature for 15 minutes. The cell-phage solution was then prepared with a trypticase soy broth agar plate containing 10 g of trypticase soy broth, 5.0 g of NaCl and 15 g of agar per liter (which was prepared at least 1 day and preheated to 37 ° C. ) Plated on. Then 10 mmol MgSO₄Overlay of agarose consisting of 0.5% agarose (ultra-pure, electrophoretic grade) dissolved in it (heated in a microwave oven until the solution boils, then cooled to 45 ° C. before use. 3-5 ml of (is) was placed on the plated cells-phage. After the agarose had solidified, the flat plate was transferred to a well-circulated ventilator at 37 ° C., where cracks were formed in the lid of the flat plate for 30 minutes, the lid was closed, and the flat plate was inverted. After 8-12 hours, plaques began to appear.

[0126] The infection resulted in total lysis of bacteria on a flat plate. Recombinant phage with HPV DNA are described by Benton, W. et al. D. Et al., Science, 196: 180 (1977), "plaque". Localized by performing "lifts)". More specifically, a flat plate of totally lysed bacteria was placed at 4 ° C. for 1 hour to solidify the agarose. An appropriately sized piece of nitrocellulose filter was then placed in contact with the center of the plate by bending in its center and by moving the center point towards the edge. Four asymmetric pores were then drilled through a nitrocellulose filter and agar with a small gauge needle, and the location of the pores was marked on the bottom of the plate with a permanent marker. This allowed the nitrocellulose filter and other regions containing a positive signal to be associated with their corresponding positions on the plate. After 10 minutes, the nitrocellulose filter is removed and the nitrocellulose filter is placed in a dish containing 200 ml 0.5 mol NaOH, 2.0 mol NaCl for 1 minute with the plaques facing upwards. By doing so, the DNA was denatured. The nitrocellulose filter was then neutralized by immersing in 500 ml of 0.5 mol Tris-HCl, 2.0 mol NaCl, pH 7.5 for 5 minutes. The filter was then rinsed in 6 × SSC consisting of 0.9 mol NaCl, 0.09 mol sodium citrate for 1 minute, dried on Watman 3MM paper and then baked under vacuum at 80 ° C. for 30 minutes.

After that, by "nick translation" as a probe³²Non-stringent hybridization using P-labeled HPV 16 DNA was performed on DNA separated from lifted plaques [see, Rigby, P. et al. J. W. Et al., Journal of Molecular Biology (J. Mol. Biol.), 113: 237 (1977) and Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982)], followed by cleaning and autoradiography. More specifically, the hybridization was carried out with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid (hereinafter "TES"). It was carried out at 41 ° C. in a solution consisting of 10 × Denhardt solution, 0.1 mmol EDTA and 10 mmol sodium phosphate (pH 7.4). Non-stringent hybridization was then carried out in 10 mmol sodium phosphate (pH 7.4), 1.1 x SSC in 0.1 mmol EDTA (0.165 mol NaCl and 0.0165 mol sodium citrate). Was performed at 52 ° C. using (consisting of).

By associating with the site of radioactive exposure, a region of the phage-containing plate containing the DNA hybridized to HPV 16 DNA was excised, and E. coli as described above. Used to reinfect coli NM538. Localization of plaques on phage containing HPV DNA was achieved by repeating the above procedure. Screened 2x10⁵Six plaques were identified from among the plaques. All clones had the same restricted map. One of the clones was selected for further study and labeled as HPV 44 clone 1.

The HPV DNA of HPV 44 clone 1 was then digested with BamHI and subcloned within a single BamHI site of pT713. The resulting recombinant DNA was labeled HPV 44 clone 2. HPV 44 Clone 2 has been added to the American Type Culture Collection at ATCC No. It was commissioned at 40353.

(B) Characterizing HPV 44 DNA 1. Hybridization studies Hybridization studies were performed on HPV 44 clone 2 DNA to prove that HPV 44 clone 2 is a new type of HPV.

More specifically, it was prepared from HPV 44 clone 2.³²P "nick-translated" DNA was hybridized to 5.0 ng of DNA from HPV types 1-43 by Southern blotting under stringent conditions. DNA from HPV types 1-43 includes Dr. Gerard Orth of the Institute Pasteur (Paris, France), the assignor of the HPV type label, and the Papilloma Reference. From Dr. Ethel-Michelle de Villiers of the Center (Pappilloma Reference Center) (Heidelberg, West Germany), and from Life Technologies, Inc. (Life) Technology, Inc. ), Obtained in a pre-immobilized form on a nitrocellulose filter. More specifically, hybridization was performed at 41 ° C. with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-Tris TES, 10 × Denhardt solution, 0.5 mmol of EDTA and 20. It was carried out in a solution consisting of mmol sodium phosphate (pH 7.4). A stringent wash was then performed at 65 ° C. with 10 mmol sodium phosphate (pH 7.4), 0.03 x SSC in 0.1 mmol EDTA (0.0045 mol NaCl and 0.00045 mol). It consisted of sodium citrate).

Significant homology was detected under non-stringent hybridization conditions between the recombinant DNA of HPV 44 clone 2 and other HPV types, but under stringent hybridization conditions the type of HPV. No homology was observed using any of 1-43, thus demonstrating that HPV 44 clone 2 represents a new type of HPV.

2. Restricted endonuclease map Figure 7 shows a restricted endonuclease map for HPV 44 clone 2. The following restriction endonucleases did not cleave HPV 44 DNA: BglI, BglII, ClaI, EcoRV, HindIII, NruI, SalI, SphI, SstI and XhoI.

3. Genome Composition To demonstrate that the genome of HPV 44 has the same or similar open reading frame to HPV 6, the following hybridization studies were performed. DNA purified from HPV 44 clone 2 as a probe under non-stringent and stringent conditions, as described above, of HPV 6 DNA.³²P "Nick-translated" fragments were used for Southern blotting. More specifically, fragments of the BamHI linearized HPV 6b clone were generated by EcoRI and PstI and then gel purified.³²Nick-translated with P and used to probe Southern blots containing the NcoI restriction digest of the purified HpaI fragment of HPV 44 clone 2 DNA. As evidenced by the results shown in FIG. 8, the DNA of HPV 44 clone 2 has the same genomic composition as HPV 6.

4. Epidemiological distribution Hybridization probes prepared from HPV 44 clone 2 efficiently hybridize only to HPV 44 DNA under stringent conditions, and these hybridization probes contain HPV 44. Used to detect genital lesions and to distinguish such genital lesions from genital lesions containing DNA of other HPV types such as 6, 11, 16, 18, 31 or 33. To prove what can be done, Washington, D.M. C. DNA from cervical biopsies and cervical swab collections containing exfoliated cells from the metropolitan area (including Mariyland and Virginia) and Michigan and surrounding states, under stringent conditions and Specific HPV using probes specific for different HPV types, including probes specific for HPV 44, by hybridization of nucleic acids under non-stringent conditions. The presence of DNA was analyzed. The biopsy was bisected, half of this sample was processed for a normal light microscope, and the other half was frozen and stored at 20 ° C. for molecular analysis. Tissues undergoing Southern blot hybridization were partitioned on cryopreservation devices to obtain material for DNA extraction. Almost all 1/18 compartments were colored with hematoxylin and eosin coloring and microscopically examined to confirm that the sample of this tissue was comparable to some of the samples analyzed by light microscopy. Exfoliated cervical cells were analyzed for paired samples by standard cytological methods, such as pap smear, the other of which was used for DNA analysis.

High molecular weight DNA was prepared from the sample as described above. 1-10 μg of purified cellular DNA was digested with PstI or BamHI, and the digested sample was electrophoresed on a 1.0% (w / v) agarose gel and transferred to a nitrocellulose filter. Hybridization was then nick-translated from the types described above under stringent conditions as described above.³²It was performed using P-labeled HPV DNA. Note: Since HPV DNA grows in pT713 or related vectors, all probes are electrophoretically purified to minimize the possibility of reaction with pT713 and related vector-like sequences in tissue samples. Most of the relevant vector sequences were removed. Additional hybridization was also performed in the presence of labeled pT713 or pBR322 and related vectors to reveal potential false positive material by plasmid-like sequences in tissue samples. The results are shown in Table 3 below. The results in Table 3 are shown graphically in FIG.

【０１３７】

[Table 3] <img file="000004.tif" id="000004" he="195" wi="135" img-format="tif" img-content="drawing" />

As evidenced by Tables 3 and 9, cervical biopsies containing HPV 44 are not only by reference to the degree of cross-hybridization in solution and by subsequent chromatography of hydroxyapatite. And also by the ability of the HPV 44 DNA probe to specifically detect and identify a well-defined population of genital lesions, i.e. those containing HPV 44 DNA as compared to those containing other types of HPV. , Which can be distinguished from biopsies containing other types of HPV, such as 6, 11, 16, 18, 31 and 33. In addition, as the results in Table 3 and FIG. 9 show, the epidemiological distribution of HPV 44 DNA between biopsies in the neck is distinguished from that found for some other HPV types.

Example 4 (A) Cloning of HPV C57 DNA The starting material used was a biopsy of vulvar condyloma from Michigan, consisting of several milligrams of tissue. The total DNA is Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982). More specifically, the tissue is subdivided and then contains 1.0 ml of 50 mmol Tris-HCl, pH 8.0 [0.6% (w / v) sodium dodecyl sulfate and 50 μg / ml Proitenase K]. Digested in 37 ° C. overnight. The resulting digest was extracted twice with 1.0 ml phenol: chloroform (1: 1 (v / v)). The DNA was then precipitated from the aqueous phase by the addition of 2 volumes of 90% (v / v) ethanol. The precipitated DNA was redissolved in 10 mmol Tris, 1.0 mmol EDTA buffer, pH 8.0 (hereinafter "TE buffer") at a concentration of about 1.0 mg / ml.

The DNA was completely digested with PstI and electrophoresed on a 1% (v / v) agarose gel, and the DNA was transferred to Southern, E. et al. M. , Journal of Molecular Biology (J. Mol. Biol. ), 98: 503 (1975), transferred to a nitrocellulose filter. The filter was then probed with DNA from HPV types 6, 11, 16, 18 and 31 under non-stringent hybridization conditions (Tm-35 ° C.) and stringent hybridization conditions (Tm-10 ° C.). Hybridization was performed at 43 ° C. with 1.0 mol of NaCl, 50 mmol of sodium phosphate buffer (pH 7.4), 1.0 mmol of EDTA, 2% (w / v) of sodium dodecyl sulfate, 0. It was performed overnight in 1% (w / v) gelatin, 50 μg / ml tRNA and 30% (w / v) formamide. Wash for 30 minutes at 55 ° C. 1.2 x SSC (1 x SSC is 0.15 mol NaCl + 0.015 mol sodium citrate), 10 mmol sodium phosphate (pH 7.4), 1.0 It was performed in mmol EDTA and 0.5% (w / v) sodium dodecyl sulfate. Hybridization was achieved with HPV types 6, 11, 16, 18 and 31 under non-stringent conditions, but not under stringent hybridization conditions.

The resulting purified DNA and λ L47 were digested with an EcoRI-restricted endonuclease. This produced a 5.1 kb fragment. This represents approximately 65% of the size of the genome of a typical papillomavirus. This fragment was cloned into a single EcoRI site at λ L47. Other parts of the other obtained purified DNA were digested with BamHI-restricted endonucleases. This produced a 5.9 kb fragment. This represents approximately 75% of the size of the genome of a typical papillomavirus. This fragment was cloned into a single EcoRI site of λL47. More specifically, 2.0 μg of the obtained purified DNA and 2.0 μg of λ L47 DNA were cleaved with 10 units of BamHI in a total volume of 50 μl of TE buffer at 37 ° C. for 1 hour. The resulting reaction mixture is then subjected to 400 μl TE. It was diluted with buffer and phenol extracted with equal volume of phenol: chloroform as described above. The aqueous phase was then extracted with chloroform: isoamyl alcohol (24: 1 (v / v)), and the DNA from the aqueous phase was precipitated with 80% (v / v) ethanol and dried. The dried DNA was then added to 10 μl of 66 mmol Tris-HCl and 6.6 mmol of MgCl.₂Suspended in 1 × ligase buffer consisting of 10 mmol DTT and 1.0 mmol ATP and incubated at 42 ° C. for 2 hours to anneal the λ arm. Next, 0.5 μl of T4 DNA ligase, about 1 unit, and 0.5 μl of 10 mmol of ATP, pH 7.0, were added to each reaction solution and binding was allowed to proceed overnight at 12 ° C. ..

Next, the binding products were packaged to form infectious phage, and E.I. Used to infect coli NM538. More specifically, E. coli growing on an agarose plate (hereinafter "TN medium") consisting of 10 g / l tryptone and 5.0 g / l NaCl. Single colonies of coli NM538 were selected and grown in 20 ml TN medium on a shaking platform (250 rpm) at 37 ° C. overnight in the early stationary phase. The cell culture was then diluted 4-fold in TN medium and grown for 3 hours. The cells are then harvested by centrifugation at 5,000 rpm for 5 minutes in a Sobalall HB-4 rotor, and the resulting cell precipitate is 10 mmol MgSO.₄It was resuspended in the original volume of 0.25 and stored at 4 ° C.

Packaged infectious phage were prepared with a commercially available BRL Lambda in vitro packaging system [see, Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)].

0.5 mol Tris-HCl (pH 8.0), 0.1 mol NaCl, 0.01 mol MgSO.₄And 1.5 × 10 in phage storage buffer (Difco) consisting of 0.01% (w / v) gelatin⁴100 μl of a suitable dilution of the packaged phage, appropriately diluted in a plaque / 9 cm diameter plate, prepared as described above in a 10-15 ml test tube. It was added to 100 μl of coli NM538, mixed gently and incubated at room temperature for 15 minutes. The cell-phage solution was then prepared with a trypticase soy broth agar plate containing 10 g of trypticase soy broth, 5.0 g of NaCl and 15 g of agar per liter (which was prepared at least 1 day and preheated to 37 ° C. ) Plated on. Then 10 mmol MgSO₄Overlay of agarose consisting of 0.5% agarose (ultra-pure, electrophoretic grade) dissolved in it (heated in a microwave oven until the solution boils, then cooled to 45 ° C. before use. 3-5 ml of (is) was placed on the plated cells-phage. After the agarose had solidified, the flat plate was transferred to a well-circulated ventilator at 37 ° C., where cracks were formed in the lid of the flat plate for 30 minutes, the lid was closed, and the flat plate was inverted. After 8-12 hours, plaques began to appear.

[0145] The infection resulted in total lysis of bacteria on a flat plate. HPV Recombinant phage with DNA are described by Benton, W. et al. D. Et al., As described in Science, 196: 180 (1977), localized by performing "plaquelifts". More specifically, a flat plate of totally lysed bacteria was placed at 4 ° C. for 1 hour to solidify the agarose. An appropriately sized piece of nitrocellulose filter was then placed in contact with the center of the plate by bending in its center and by moving the center point towards the edge. Four asymmetric pores were then drilled through a nitrocellulose filter and agar with a small gauge needle, and the location of the pores was marked on the bottom of the plate with a permanent marker. This allowed the nitrocellulose filter and other regions containing a positive signal to be associated with their corresponding positions on the plate. After 10 minutes, the nitrocellulose filter is removed and the nitrocellulose filter is placed in a dish containing 200 ml 0.5 mol NaOH, 2.0 mol NaCl for 1 minute with the plaques facing upwards. By doing so, the DNA was denatured. The nitrocellulose filter was then neutralized by immersing in 500 ml of 0.5 mol Tris-HCl, 2.0 mol NaCl, pH 7.5 for 5 minutes. The filter was then rinsed in 6 × SSC consisting of 0.9 mol NaCl, 0.09 mol sodium citrate for 1 minute, dried on Watman 3MM paper and then baked under vacuum at 80 ° C. for 30 minutes.

[0146] Then, as a probe, by "nick translation"³²Non-stringent hybridization using P-labeled HPV 16 DNA was performed on DNA separated from lifted plaques [see, Rigby, P. et al. J. W. Et al., Journal of Molecular Biology (J. Mol. Biol.), 113: 237 (1977) and Maniatis, T. et al. Et al., Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Laboratories), Cold Spring Harbor, NY (1982)], followed by cleaning and autoradiography. More specifically, the hybridization was carried out with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid (hereinafter "TES"). It was carried out at 41 ° C. in a solution consisting of 10 × Denhardt solution, 0.1 mmol EDTA and 10 mmol sodium phosphate (pH 7.4). Non-stringent hybridization was then performed in 10 mmol sodium phosphate (pH 7.4), 1.1 x SSC in 0.1 mmol EDTA (0.165 mol NaCl and 0.0165 mol sodium citrate). or Ranaru) was carried out at 52 ° C. using.

By associating with the site of radioactive exposure, a region of the phage-containing plate containing the DNA hybridized to HPV 16 DNA was excised, and E. coli as described above. Used to reinfect coli NM538. Localization of plaques on phage containing HPV DNA was achieved by repeating the above procedure. 1.5x10 screened from cloning using EcoRI digested DNA⁵Two plaques were identified from among the plaques. Both clones had the same restricted map. One of the clones was selected for further study and labeled as HPV C57 Clone 1A. One clone was screened from cloning using BamHI digested DNA 5x10⁴Was identified from among the plaques of HPV C57 clone 1B. Identified. The cloned fragment showed a size of 2.85 kb and was labeled HPV C57 Clone 1B.

HPV C57 clones 1A and 1B of HPV DNA were then digested with EcoRI or BamHI, respectively, and subcloned into a single EcoRI or BamHI site of pT713. The resulting recombinant DNA was labeled HPVC57 clones 2A and 2B. HPV C57 clones 2A and 2B are included in the American Type Culture Collection, respectively, at ATCC No. 40341 and ATCC No. It was commissioned at 40379.

(B) Characterizing HPV C57 DNA 1. Hybridization studies Hybridization studies were performed on HPV C57 clones 2A and 2B DNA to show that HPV C57 clones 2A and 2B are new types of HPV. Proven.

More specifically, it was prepared from HPV C57 clones 2A and 2B.³²P "nick-translated" DNA was hybridized to 5.0 ng of DNA from HPV types 1-45 by Southern blotting under stringent conditions. DNA from HPV types 1-45 is available from Dr. Gerard Orth of the Institute Pasteur (Paris, France), the transferor of HPV type labeling, the Papilloma Reference Center. Preliminary on a nitrocellulose filter from Dr. Ethel-Michelle de Villiers of (Pappilloma Reference Center) (Heidelberg, West Germany) and from Life Technologies, Inc. Obtained in a fixed form. More specifically, hybridization was performed at 41 ° C. with 1.0 mol of NaCl, 28% (v / v) formamide, 50 mmol of N-tris. It was carried out in a solution consisting of TES, 10 × Denhardt solution, 0.5 mmol of EDTA and 20 mmol of sodium phosphate (pH 7.4). A stringent wash was then performed at 65 ° C. with 10 mmol sodium phosphate (pH 7.4), 0.03 x SSC in 0.1 mmol EDTA (0.0045 mol NaCl and 0.00045 mol). It consisted of sodium citrate).

Significant homology was detected under non-stringent hybridization conditions between the recombinant DNA of HPV C57 clones 2A and 2B and other HPV types, but under stringent hybridization conditions HPV. No homology was observed using any of types 1-45, thus demonstrating that HPVC57 clones 2A and 2B represent the new type of HPV.

2. Restricted endonuclease map Figure 10 shows a restricted endonuclease map for HPV C57 clones 2A and 2B. The following restriction endonucleases did not cleave HPV C57 DNA: BglII, NcoI, HindIII, SalI, SstI and XbaI.

3. Genome Composition The following hybridization studies were performed to demonstrate that the genome of HPV C57 has the same or similar open reading frame to HPV 6. DNA purified from HPV C57 clones 2A and 2B was used as a probe under non-stringent and stringent conditions as described above for HPV 6 DNA.³²P "Nick-translated" fragments were used for Southern blotting. More specifically, fragments of the BamHI linearized HPV 6b clone were generated by EcoRI and PstI and then gel purified.³²Nick-translated with P and used to probe Southern blots containing the purified EcoRI + EcoRV restriction digest of HPV C57 clone 2A DNA and the EcoRI + EcoRV digest of the purified BamHI fragment of HPV C57 clone 2A DNA. As evidenced by the results shown in FIG. 11, the DNA of HPV C57 clones 2A and 2B has the same genomic composition as HPV 6.

4. Epidemiological distribution Hybridization probes prepared from HPV C57 clones 2A and 2B efficiently hybridize only to HPV C57 DNA under stringent conditions, and these hybridization probes are HPV C57. To detect genital lesions containing genital lesions and to distinguish such genital lesions from genital lesions containing DNA of other HPV types such as 6, 11, 16, 18, 31 or 33. To prove that it can be used in Washington, D.C. C. DNA from cervical biopsies and cervical swab collections containing exfoliated cells from the metropolitan area (including Mariyland and Virginia) and Michigan and surrounding states, under stringent conditions and Specific HPV using probes specific for different HPV types, including probes specific for HPV C57 by hybridization of nucleic acids under non-stringent conditions. The presence of DNA was analyzed. The biopsy was bisected, half of this sample was processed for a normal light microscope, and the other half was frozen and stored at 20 ° C. for molecular analysis. Tissues undergoing Southern blot hybridization were partitioned on cryopreservation devices to obtain material for DNA extraction. Almost all 1/18 compartments were colored with hematoxylin and eosin coloring and microscopically examined to confirm that the sample of this tissue was comparable to some of the samples analyzed by light microscopy. Exfoliated cervical cells were analyzed for paired samples by standard cytological methods, such as pap smear, the other of which was used for DNA analysis.

High molecular weight DNA was prepared from the sample as described above. 1-10 μg of purified cellular DNA was digested with PstI or BamHI, and the digested sample was electrophoresed on a 1.0% (w / v) agarose gel and transferred to a nitrocellulose filter. Hybridization was then nick-translated from the types described above under stringent conditions as described above.³²It was performed using P-labeled HPV DNA. (For HPV C57 DNA, a mixture of HPV DNA from HPV C57 clones 2A and 2B was used.) Note: HPV DNA grows in pT713 or related vectors, so pT713 and related vector-like sequences in tissue samples. To minimize the possibility of reaction with, all probes were electrophoretically purified to remove most of the vector sequences involved. Additional hybridization was also performed in the presence of labeled pT713 or pBR322 and related vectors to reveal potential false positive material by plasmid-like sequences in tissue samples. The results are shown in Table 4 below. The results in Table 4 are shown graphically in FIG.

【０１５６】

[Table 4] <img file="000005.tif" id="000005" he="195" wi="135" img-format="tif" img-content="drawing" />

As evidenced by Tables 4 and 12, cervical biopsies containing HPV C57 are not only by reference to the degree of cross-hybridization in solution and by subsequent chromatography of hydroxyapatite. And also the ability of the HPV C57 DNA probe to specifically detect and identify a well-defined population of genital lesions, i.e. those containing HPV C57 DNA as compared to those containing other types of HPV. Can be distinguished from biopsies containing other types of HPV, such as 6, 11, 16, 18, 31 and 33. In addition, as the results in Table 4 and FIG. 12 show, the epidemiological distribution of HPVC57 DNA between biopsies in the neck is distinguished from that found for some other HPV types.

Although the present invention has been described in detail and with respect to specific embodiments, it is clear that various modifications and modifications are possible without departing from the spirit and scope of the invention.

[0159] The main features and aspects of the present invention are as follows.

[0160] 1. Cloning vectors and substantially all of HPV 35 DNA or fragments thereof, substantially all of HPV 43 DNA or fragments thereof, substantially all of HPV 44 DNA or fragments thereof, or HPV C57 DNA or fragments thereof. Recombinant DNA of HPV 35, HPV 43, HPV 44 or HPV C 57, characterized in that it comprises substantially all.

[0161] 2. The recombinant DNA according to the above item 1, wherein the cloning vector is selected from the group consisting of pBR322, pUC11, λSharon, λL47, M13-induced bacteriophage, pZIP-Neo SV [X1], pBKTK-1, pT712 and pT713. ..

[0162] 3. The recombinant DNA according to the above item 1, wherein the fragment has a size of about 15 to about 8000 base pairs.

4. The recombinant DNA according to the third item, wherein the fragment has a size of about 300 to about 800 base pairs.

5. The recombinant DNAs are HPV 35 clones 2A and 2B (ATCC No. 40330 and ATCC No. 40331, respectively), HPV 43 clones 2A and 2B (ATCC No. 40338 and ATCC No. 40339, respectively), and HPV 44 clones (ATCC). No. 40353) or HPV C57 clones 2A and 2B (ATCC No. 40341 and ATCC No. 40379, respectively).

6. The recombinant DNA according to the above item 1, wherein the HPV DNA of the recombinant DNA is labeled with a marker.

7. The recombinant DNA according to the above item 6, wherein the marker is a radioactive marker.

8. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The recombinant DNA according to the above item 7, which is a radioactive marker selected from S.

9. The recombinant DNA according to the above item 6, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, an enzyme and a fluorescent molecule.

10. 69. The recombinant DNA according to the above item 9, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase. 11. The recombinant DNA according to the above item 9, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

12. Essentially pure HPV 35 DNA or a fragment thereof, essentially pure HPV 43 DNA or a fragment thereof, essentially pure HPV 44 DNA or a fragment thereof, or essentially pure HPV C57 DNA or a fragment thereof.

13. The HPV DNA or a fragment thereof is an essentially pure HPV DNA according to the above item 12, which is biologically produced.

14. The HPV DNA or a fragment thereof is chemically produced as described in the above item 12, which is essentially pure HPV DNA.

15.73. The essentially pure HPV DNA according to item 12 above, wherein the fragment is about 15 to about 8000 bases or base pair in size.

16. The essentially pure HPV DNA according to item 15 above, wherein the fragment is about 300 to about 800 bases or base pair size.

17.75. The essentially pure HPV DNA according to item 12 above, wherein the HPV DNA or a fragment thereof is labeled with a marker.

18.76. The essentially pure HPV DNA according to item 17 above, wherein the marker is a radioactive marker.

19. 19. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The essentially pure HPV DNA according to paragraph 18 above, which is a radioactive marker selected from S.

20. The essentially pure HPV DNA according to item 17 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

21. The essentially pure HPV DNA according to item 20 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

22. The essentially pure HPV DNA according to item 20 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

23. Essentially pure HPV 35 RNA or fragments thereof, essentially pure HPV 43 RNA or fragments thereof, essentially pure HPV 44 RNA or fragments thereof, or essentially pure HPV C57 RNA or fragments thereof.

24. The HPV RNA or fragment thereof is a biologically produced essentially pure HPV RNA according to item 23 above.

25. The essentially pure HPV RNA according to item 23 above, wherein the HPV RNA or fragment thereof is chemically produced.

26. The essentially pure HPV RNA according to item 23 above, wherein the fragment is about 15 to about 8000 bases or base pair in size.

27. The essentially pure HPV RNA according to item 26 above, wherein the fragment is about 300 to about 800 bases or base pair size.

28. The essentially pure HPV RNA according to item 23 above, wherein the HPV RNA or fragment thereof is labeled with a marker.

29. The essentially pure HPV RNA according to item 28 above, wherein the marker is a radioactive marker.

30. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The essentially pure HPV RNA according to paragraph 29 above, which is a radioactive marker selected from S.

31. The essentially pure HPV RNA according to item 28 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

32. The essentially pure HPV RNA according to item 31 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

33. The essentially pure HPV RNA according to item 31 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

34. (I) Marker-labeled HPV 35 DNA or fragment thereof, (ii) Marker-labeled HPV 35 RNA or fragment thereof, (iii) Marker-labeled HPV 43 DNA or fragment thereof, (iv) Marker. Labeled HPV 43 RNA or fragment thereof, (v) Marker-labeled HPV 44 DNA or fragment thereof, (vi) Marker-labeled HPV 44 RNA or fragment thereof, (vii) Marker-labeled HPV C57 DNA Alternatively, an HPV hybridization probe selected from the group consisting of a fragment thereof and HPV C57 RNA labeled with a (viii) marker or a fragment thereof.

35. The HPV hybridization probe according to item 34 above, wherein the fragment has a size of about 15 to about 8000 bases or base pairs.

36. The HPV hybridization probe according to item 35 above, wherein the fragment has a size of about 300 to about 800 bases or base pairs.

37. The HPV hybridization probe according to the above item 34, wherein the marker is a radioactive marker.

38. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The HPV hybridization probe according to item 37 above, which is a radiomarker selected from S.

39. The HPV hybridization probe according to item 34 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

40. The HPV hybridization probe according to item 39 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

41. The HPV hybridization probe according to item 39 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

42. Members selected from the group consisting of (a) (i) marker-labeled HPV 35 DNA or fragments thereof and (ii) marker-labeled HPV 35 RNA or fragments thereof, and (b) labeled with markers. At least one other HPV type DNA or RNA or fragment thereof, or (a') (i) marker-labeled HPV 43 DNA or fragment thereof and (ii) marker-labeled HPV 43 RNA or fragment thereof. A member selected from the group consisting of, and at least one other HPV type DNA or RNA or fragment thereof labeled with (b') marker, or HPV 44 labeled with (a ") (i) marker. A member selected from the group consisting of DNA or fragments thereof and (ii) marker-labeled HPV 44 RNA or fragments thereof, and (b ") marker-labeled at least one other HPV type DNA or RNA. Or a fragment thereof, or (a "') (i) marker-labeled HPV C57 DNA or a fragment thereof and (ii) marker-labeled HPV C57. A member selected from the group consisting of RNA or fragments thereof, and an HPV hybridization probe selected from the group consisting of at least one other HPV type DNA or RNA labeled with the (b "') marker or fragments thereof. Composition.

43. The HPV hybridization probe composition according to item 42 above, wherein the fragment has a size of about 15 to about 8000 bases or base pairs.

44. The HPV hybridization probe composition according to item 43 above, wherein the fragment has a size of about 300 to about 800 bases or base pairs.

45. The HPV hybridization probe composition according to the above item 42, wherein the marker is a radioactive marker.

46. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The HPV hybridization probe composition according to the above item 45, which is a radioactive marker selected from S.

47. The HPV hybridization probe composition according to item 42 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

48. The HPV hybridization probe composition according to item 47 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

49. The HPV hybridization probe composition according to item 47 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

50. The hybridization probe composition according to item 42 above, wherein the other HVP type is at least one member selected from the group consisting of HVP 6, HVP 11, HVP 16, HVP 18 and HVP 31.

51. The hybridization probe composition according to the above item 50, wherein the other HVP type is a member of at least one selected from the group consisting of HVP 16, HVP 18 and HVP 31.

52. The hybridization probe composition according to item 51 above, wherein the other HVP types are HVP 16, HVP 18, and HVP 31.

53. Steps: (1) Under non-stringent conditions, (a) (i) marker-labeled HPV 35 DNA or fragment thereof, marker-labeled HPV 43 DNA or fragment thereof, marker-labeled HPV 44 DNA. Or fragments thereof, or marker-labeled HPVC 57 DNA or fragments thereof, and (ii) marker-labeled HPV 35 RNA or fragments thereof, marker-labeled HPV 43 RNA or fragments thereof, marker-labeled HPV Hybridization was performed using a member selected from the group consisting of 44 RNA or fragments thereof, or marker-labeled HPV C57 RNA or fragments thereof, and (b) an unknown sample of DNA or RNA. , And (2) a method for detecting DNA or RNA of HVP comprising, assaying for the presence of cross-hybridation to detect DNA or RNA of HVP in said sample.

54. 53. The method of paragraph 53 above, wherein the unknown sample of DNA or RNA is derived from a lesion in the genitals, throat, mouth or skin.

55. 58. The method of item 54 above, wherein the unknown sample of DNA or RNA is derived from a lesion of the genitals.

56. The unknown sample of DNA or RNA from a genital lesion was obtained by obtaining cells stripped by biopsy of the epithelial lesion, cervical rubbing, or cervical swabbing, or cloned in a cloning vector. 55. The method of item 55 above, which is DNA derived from a genital lesion.

57. 5. The method of item 53 above, wherein the fragment is about 15 to about 8000 bases or base pair in size.

58. 58. The method of item 57 above, wherein the fragment is about 300 to about 800 bases or base pair in size.

59. Hybridization is also selected from the group consisting of (c) (i) marker-labeled HPV 6 DNA or fragments thereof and (ii) marker-labeled HPV 6 RNA or fragments thereof, (i). A member selected from the group consisting of) marker-labeled HPV 11 DNA or fragments thereof and (ii) marker-labeled HPV 11 RNA or fragments thereof, (i) marker-labeled HPV 16 DNA or fragments thereof. A member selected from the fragment and the group consisting of (ii) marker-labeled HPV 16 RNA or fragments thereof, and (i) marker-labeled HPV 18 DNA or fragments thereof and (ii) marker-labeled. 53. The method of paragraph 53 above, performed using at least one of a member selected from the group consisting of HPV 18 RNA or fragments thereof.

60. 5. The method of paragraph 59 above, wherein the other type is a member of at least one selected from the group consisting of HVP 16 and HVP 18.

61. The method according to paragraph 60 above, wherein the other HVP types are HVP 16 and HVP 18.

62. The method according to paragraph 53 above, wherein the marker is a radioactive marker.

63. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The method according to paragraph 62 above, which is a radioactive marker selected from S.

64. 5. The method of item 53 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

65. The method according to paragraph 64 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

66. 6. The method of item 64 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

67. The method of item 53 above, wherein the cross-hybridization produces a DNA-DNA hybrid.

68. The method of item 53 above, wherein the cross-hybridization produces a DNA-RNA hybrid.

69. Steps: (1) Under stringent conditions, (a) and (i) marker-labeled HPV 35 DNA or fragments thereof, marker-labeled HPV 43 DNA or fragments thereof, marker-labeled HPV, respectively. 44 DNA or fragments thereof, or marker-labeled HPV C57 DNA or fragments thereof, and (ii) marker-labeled HPV 35 RNA or fragments thereof, marker-labeled HPV 43 RNA or fragments thereof, markers, respectively. Using a member selected from the group consisting of HPV 44 RNA or fragments labeled with, or marker-labeled HPV C57 RNA or fragments thereof, and (b) an unknown sample of DNA or RNA. Hybridization is performed and (2) assayed for the presence of cross-hybridation, respectively, in the sample, HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPV C57 DNA or HPV 35, which comprises detecting RNA. A method for detecting DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPVC 57 DNA or RNA.

70. 6. The method of paragraph 69 above, wherein the unknown sample of DNA or RNA is derived from a lesion in the genitals, throat, mouth or skin.

71. The method of item 70 above, wherein the unknown sample of DNA or RNA is derived from a lesion of the genitals.

72. The unknown sample of DNA or RNA from a genital lesion was obtained by obtaining cells stripped by biopsy of the epithelial lesion, cervical rubbing, or cervical swabbing, or cloned in a cloning vector. The method according to paragraph 71 above, which is DNA derived from a genital lesion.

73. 6. The method of item 69 above, wherein the fragment is about 15 to about 8000 bases or base pair in size.

74. The method according to item 73 above, wherein the fragment is about 300 to about 800 bases or a base pair size.

75. HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA are substantially all of the HPV 35 genome, substantially all of the HPV 43 genome, substantially all of the HPV 44 genome, or 6. The method of paragraph 69 above, which comprises substantially the entire genome of HPV C57.

76. The method according to item 69 above, wherein the marker is a radioactive marker.

77. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The method according to paragraph 76 above, which is a radioactive marker selected from S.

78. 6. The method of item 69 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

79. The method of item 78 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

80. The method of item 78 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

81. The method of item 69 above, wherein the cross-hybridization produces a DNA-DNA hybrid.

82. The method of item 69 above, wherein the cross-hybridization produces a DNA-RNA hybrid.

83. Steps: (1) Under stringent conditions, (a) Sampling of genital lesions First fraction of DNA or RNA derived from each genital lesion, said sampling of epidemiological progression to cervical cancer Shown and (b) (i), respectively, marker-labeled HPV 35 DNA or fragment thereof, marker-labeled HPV 43 DNA or fragment thereof, marker-labeled HPV 44 DNA or fragment thereof, or marker. HPV C57 DNA or fragment thereof labeled with, and (ii) marker-labeled HPV 35 RNA or fragment thereof, marker-labeled HPV 43 RNA or fragment thereof, marker-labeled HPV 44 RNA or Hybridization was performed using the fragment, or a member selected from the group consisting of marker-labeled HPV C57 RNA or a fragment thereof, and (2) under stringent conditions, (a). A second fraction of DNA or RNA derived from each genital lesion in the sampling of the genital lesion, and Hybridization was performed using (b) an unknown sample of DNA or RNA derived from marker-labeled genital lesions, and (3) epidemics of cross-hybridation obtained in step (1). HPV 35 DNA or RNA, HPV 43 DNA in the sample, respectively, comparing the target distribution with that obtained in step (2) and cross-hybridation of DNA from each lesion constituting the epidemiological distribution. Alternatively, it comprises detecting RNA, HPV 44 DNA or RNA, or HPV C57 DNA or RNA, HPV 35 DNA or RNA, HPV 43 DNA or RNA, HPV 44 DNA or RNA, or HPVC 57 DNA or. A method for detecting RNA.

84. The method of item 83 above, wherein the fragment is about 15 to about 8000 bases or base pair in size.

[0243] 85. 8. The method of item 84 above, wherein the fragment is about 300 to about 800 bases or base pair in size.

86. HPV 35 DNA, HPV 43 DNA, HPV 44 DNA or HPV C57 DNA are substantially all of the HPV 35 genome, substantially all of the HPV 43 genome, substantially all of the HPV 44 genome, or The method according to paragraph 83 above, which comprises substantially the entire genome of HPV C57.

87. The method according to item 83 above, wherein the marker is a radioactive marker.

88. The marker is³²P,¹⁴C,³H,¹²⁵I and³⁵The method according to item 87 above, which is a radioactive marker selected from S.

89. 8. The method of item 83 above, wherein the marker is a non-radioactive marker selected from the group consisting of biotin, enzymes and fluorescent molecules.

90. 8. The method of item 89 above, wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

91. 8. The method of item 89 above, wherein the fluorescent molecule is selected from the group consisting of fluorescein or rhodamine.

92. The unknown sample of DNA or RNA from a genital lesion was obtained by obtaining cells stripped by biopsy of the epithelial lesion, cervical rubbing, or cervical swabbing, or cloned in a cloning vector. 8. The method of item 83 above, which is DNA derived from a genital lesion.

93. The method of item 83 above, wherein the cross-hybridization produces a DNA-DNA hybrid.

94. The method of item 83 above, wherein the cross-hybridization produces a DNA-RNA hybrid.

[Simple explanation of drawings]

FIG. 1 shows a restricted nuclease map of HPV 35 DNA. The following codes are used to represent restriction enzyme sites: B1, BamHI; E5, EcoRV; H3, HindIII; P1, PstI; P2, PstII; and S1, SphI.

FIG. 2 shows a region of partial homology between HVP 6 and HPV 35 DNA as determined by hybridization of nucleic acids under non-stringent hybridization conditions. Arrows connect regions that indicate homology. Dashed arrows indicate regions with only weak homology. The smallest BamHI-PstI fragment of HVP 6 did not hybridize to HPV 35. Each of the maps in FIG. 1 is arranged so that the end of the straight map corresponds to the relative position of the HpaI site of HVP 6. The estimated open frame positions for HVP 6 are shown above the homology map.

FIG. 3 graphically shows the distribution of HPV types in various lesions based on Table 1.

FIG. 4 shows a restricted nuclease map of HPV 43 DNA.

FIG. 5 shows a region of partial homology between HVP 6 and HPV 43 DNA as determined by hybridization of nucleic acids under non-stringent hybridization conditions. Arrows connect regions that indicate homology. Each of the maps in FIG. 5 is arranged so that the end of the straight map corresponds to the relative position of the HpaI site of HVP 6. The estimated open frame positions for HVP 6 are shown above the homology map.

FIG. 6 graphically shows the distribution of HPV types in various lesions based on Table 2.

FIG. 7 shows a restricted nuclease map of HPV 44 DNA.

FIG. 8 shows a region of partial homology between HVP 6 and HPV 44 DNA as determined by hybridization of nucleic acids under non-stringent hybridization conditions. Arrows connect regions that indicate homology. Dashed arrows indicate regions with only weak homology. Each of the maps in FIG. 8 is arranged so that the end of the straight map corresponds to the relative position of the HpaI site of HVP 6. The estimated open frame positions for HVP6 are shown above the homology map.

FIG. 9 graphically shows the distribution of HPV types in various lesions based on Table 3.

FIG. 10 shows a restricted nuclease map of HPV C57 DNA.

FIG. 11 shows a region of partial homology between HVP 6 and HPV C57 DNA as determined by hybridization of nucleic acids under non-stringent hybridization conditions. Arrows connect regions that indicate homology. Each of the maps in FIG. 11 is arranged so that the end of the straight map corresponds to the relative position of the HpaI site of HVP 6. The estimated open frame positions for HVP 6 are shown above the homology map.

FIG. 12 graphically shows the distribution of HPV types in various lesions based on Table 4.

Continuation of front page (31) Priority claim number 060883 (32) Priority date June 12, 1987 (June 12, 1987) (33) Priority claim country United States (US) (31) Priority Claim number 114985 (32) Priority date October 30, 1987 (October 30, 1987) (33) Priority claiming country United States (US) Microbial contract number ATCC 40338 Microbial contract number ATCC 40339 (58) Investigated Field (Int.Cl.⁷, DB name) C12N 15/00 --15 / 90 C12Q 1/68 BIOSIS (DIALOG) WPI (DIALOG)