WO2015010347A1 - 人乳头瘤病毒e6蛋白可诱发同源蛋白间交叉反应性抗体的精细表位肽 - Google Patents

人乳头瘤病毒e6蛋白可诱发同源蛋白间交叉反应性抗体的精细表位肽 Download PDF

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WO2015010347A1
WO2015010347A1 PCT/CN2013/080798 CN2013080798W WO2015010347A1 WO 2015010347 A1 WO2015010347 A1 WO 2015010347A1 CN 2013080798 W CN2013080798 W CN 2013080798W WO 2015010347 A1 WO2015010347 A1 WO 2015010347A1
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protein
epitope
human papillomavirus
polypeptide
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刘日廷
徐万祥
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Liu Riting
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • Human papillomavirus E6 protein induces homologous protein
  • the invention belongs to the technical field of immunology and biomedicine, and particularly relates to a minimal motif peptide which can be recognized by the monoclonal antibody C 1P5 in human papillomavirus E6 protein and application thereof. Background technique
  • Cervical cancer is the second largest cancer in the world that causes women to die, and its threat is second only to breast cancer. About 500,000 people worldwide are diagnosed with cervical cancer each year, and about 300,000 people die of this cancer. The annual incidence rate in China is estimated at 135,000 and the death toll is 50,000.
  • HPV human papillomavirus
  • cervical cancer was determined in the early 1980s. The latest data indicates that HPV can be detected in 99.8% of cervical cancer biopsy specimens. Therefore, attention to women's reproductive health, researchers in various countries have long been researching ways to diagnose, prevent and treat HPV. Excitingly, in 2006 and 2007, two preventive tetravalent and bivalent HPV vaccines were first developed by Merck and GSK Pharmaceuticals.
  • HPV prevention and treatment there are still many issues that need to be overcome by humans.
  • HPV infecting epithelial tissues such as the female genital tract
  • HPVs that can cause cervical cancer through persistent infection
  • HPV therapeutic peptide vaccines have selected cytotoxic T cell epitopes (TCL/TcCE) peptides on HPV 16 or 18 E6 and E7 proteins, they have also been developed to target early oncogenic E6 and E7 proteins, while inducing CD4+.
  • Therapeutic HPV fusion protein vaccines for CD8+ and antibody responses suggest that prophylactic HPV multi-epitope peptide vaccines that combine their B- and T-cell epitopes can also be developed, ie by generating humoral responses to antibodies, as well as in precancerous lesions. Or to exclude the tumor-specific immune response before cervical malignant lesions to achieve full or partial clearance of HPV.
  • the reality is that the practical application of serological tests or Kits in clinical tests has not been seen so far, because it is difficult to rule out the high proportion of false positive results that are common in ELISA tests but are not expected by both doctors and patients. It is conceivable that the false positive result is formed by the combination of the following three factors, namely: 1) The antibody titer in response to HPV infection in the general patient is usually not high, so the dilution of the serum sample is not high (commonly used 1: 2 : 1 : 10 serum dilution); 2) due to anti-virus and other microbial infections and autoimmune factors, there are often thousands of countless anti-unantigen antigen antibodies in the human body; 3) using 18-20 constituents to detect single antibodies The epitope peptide is used as a detection antigen [Dillner J, Dillner L, Cheng HM.
  • Synthetic peptides in human papillomavirus 1, 5, 6, 8, 1 1, 16, 18, 31, 33 and 56, useful in immunoassay for diagnostic purposes United States Patent Number: 5932412, 1999]
  • linear B cell epitopes usually consist of 3-8 residues, so that epitope peptides larger than 8 residues are apparently potentially N recognized by unknown antigen antibodies in vivo.
  • Epitope the probability of being recognized by one or several antibodies in the body in the presence of low serum dilution.
  • the same low titer non-target antibody in the body drops below the detection sensitivity as the serum dilution increases, thereby reducing the possible cross-reactivity and avoiding false positive results.
  • the realization of the above target for the detection of antigen Kit depends on the identification of more linear epitopes of the target antigen and its minimal motif.
  • an antigenic epitope represents an antibody
  • an antibody corresponds to an epitope
  • the potential application of epitopes and antibodies in antiviral antitumor drugs (vaccines) and diagnostic reagents and thus the identification of unquestionable epitopes
  • the preparation has intellectual property rights for invention and preparation.
  • the formation of intellectual property in this area is also due to the difficulty of identifying or preparing the technology.
  • Banks L et al. used recombinant HPV18 E6 protein to prepare six monoclonal antibodies including C 1P5 [Banks L, Spence P, Androphy E, et al.
  • X is selected from the group consisting of amino acids L or Y;
  • polypeptide is derived from the E6 protein of human papillomavirus (HPV).
  • polypeptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the polypeptide is derived from human papillomavirus type 18 (X is L), type 16 (X is Y), type 52 (X is ⁇ ), type 33 (X is ⁇ ) or 58.
  • Type (X is ⁇ ) ⁇ 6 protein in another aspect of the invention, there is provided the use of a polypeptide as described above for the preparation of an immunogen against a human papillomavirus ⁇ 6 protein.
  • the human papillomavirus is of the 18, 16, 52, 33 or 58 type.
  • the use of the polypeptide for the preparation of a multi-epitope peptide detection antigen for serological diagnosis of HPV18 infection either alone or in combination.
  • the use of the polypeptides described above is provided for use as an epitope marker for the identification of HPV 18, 16, 52, 33 or 58 infections based on their encoded amino acid sequence after PCR amplification of the ⁇ 6 gene.
  • the use of the monoclonal antibody C1P5 for the preparation of a human nipple is provided A kit for a tumor virus, wherein the human papillomavirus is a virus of type 58, 33 or 52.
  • kits for detecting human papillomavirus wherein the human papillomavirus is a virus of type 58, 33 or 52, and the kit comprises a monoclonal antibody C1P5.
  • the kit comprises a monoclonal antibody C1P5.
  • FIG. 1 Conservative analysis of the HPV58-E6 protein E6-2 epitope motif in high-risk HPV homologous proteins.
  • the HPH58 E6-2 motif YGDTL in the box is 100% compliant with the corresponding sequence in the HPV18-E6 protein.
  • Other high-risk HPV homologous protein sequences were aligned, and there was one residue difference at the same position, but the octapeptides expressed by their fusion with truncated GST188 were recognized by the anti-recombinant HPV58-E6 protein polyclonal antibody.
  • the epitope motif similarity is calculated based on the 5 residues in the box.
  • HPV18-E6 mAb C1P5 may identify HPV homologous protein sequences for epitope motifs.
  • an immunogen against HPV E6 protein can be prepared using a polypeptide containing the epitope (also referred to as a short peptide or a motif peptide in the present invention) (in combination with other epitopes)
  • the multi-epitope peptide immunogen is constructed, or an antibody specific for the HPV E6 protein is prepared, and the obtained immunogen or antibody can be used for the prevention and diagnosis of various HPV subtypes.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • E6 protein refers to a protein present in HPV, and in the present invention, the E6 protein-encoding gene sequence and the protein primary structure sequence of HPV16, HPV18, HPV33, HPV52 and HPV58 are respectively according to their standard strains.
  • GenBank accession numbers are: NC—001526, NC—001357, EU918766, X74481 and D90400.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly constituted by”, “consisting essentially of” and “consisting of” belonging to “contains” , the subordinate concept of "having” or “including”.
  • specificity of an antibody refers to an antibody that binds to a particular subtype of HPV E6 protein or fragment. Specifically, those antibodies that bind to a specific subtype of HPV E6 protein. Epitope and its use
  • the inventors analyzed a commercial HPV18/E6 mAb (C1P5 mAb) to determine whether it recognizes the HPV58/E6-2 epitope, and thus determined that the murine monoclonal antibody C1P5 recognizes HPV18 E6. The fine base of the /2 epitope.
  • the inventors unexpectedly found that the C1P5 mAb produced an immunoblot reaction with the recombinant HPV58 type E6 protein, but did not recognize the YGDTL-containing motif octapeptide.
  • a unique strategy is used to carry out related epitope identification studies, which ultimately form the technical solution of the present invention.
  • HPV18-E6 protein C1P5 monoclonal antibody can be immunoblot cross-reactive with the recombinant HPV58-E6 protein, but does not recognize the HPV58-containing E6-2 minimal motif which is highly conserved in the homologous protein.
  • YGDTL highly conserved in homologous proteins, Figure 1
  • octapeptide fusion protein octapeptide fusion protein.
  • C1P5 monoclonal antibody recognizes LRLLSKISEYRHYNY and ISEYRHYNY SLYGDT with 15 residues of 15 residues overlapping each other, that is, The mAb recognition motif falls within the 9 residue overlap region (ISEYRHYNY).
  • C1P5 monoclonal antibody The other minimal motifs are further reduced to the ELRHY pentapeptide motif with only one residue difference between each other, because if one residue is extended left or left to form a hexapeptide motif, then the three protein sequences are It is impossible to recognize C1P5 mAb by displaying 2 residue differences.
  • a short peptide fusion protein expressing HPV18 P2-P5 and HPV16/18 P6 was reconstituted (Fig. 3A).
  • the immunoblotting assay only the P5 and P6 short peptides produced a reactive band (Fig. 3B), thus confirming that the C1P5 mAb recognizes the HPV18-E6 protein with the smallest epitope of the ELRHY pentapeptide and is a specific epitope of HPV18.
  • the motif (Fig. 2) and a conserved EYRHY pentapeptide sequence that cross-reacted with HPV class 16/58 was confirmed (Figs.
  • HPV18 C1P5 monoclonal antibody was found to cross-react with high-risk HPV33 and HPV52 E6 proteins, and its sequence was also reactive with HPV16/58.
  • the peptides are identically conserved sequences ( Figure 2).
  • the polypeptide has the amino acid sequence shown by SEQ ID NO: 1 (ELRHY) or SEQ ID NO: 2 (EYRHY).
  • the polypeptide fusion protein can be obtained in large quantities by recombinant methods. This is usually carried out by cloning into a vector, transferring it to a cell, and then isolating the fusion protein of the relevant sequence from the proliferated host cell by a conventional method. Further, in comparison with the short peptide of the present invention, it is preferred to synthesize the relevant sequence by a method of artificial synthesis (e.g., synthesis by a polypeptide synthesizer), and the method of artificial synthesis can obtain the desired polypeptide simply and quickly.
  • a method of artificial synthesis e.g., synthesis by a polypeptide synthesizer
  • the invention also provides for the use of the identified epitope minimal motif peptides and their extended peptides in the preparation of "universal" prophylactic/therapeutic HPV recombinant multi-epitope peptide vaccines.
  • the minimal motif peptide of the present invention can be used as a potent antigenic fragment of the E6 protein for the preparation of universal antibodies that specifically bind to the E6 protein, including monoclonal or polyclonal antibodies.
  • the minimal motif peptides of the invention may also be linked to other peptides or immunologically acceptable carriers, preferably which are themselves immunogenic, for use in the preparation of antibodies as immunogens.
  • Antibodies can be prepared by a variety of techniques known to those skilled in the art. For example, a purified polypeptide of the invention (or after attachment to other peptides or an immunologically intractable vector) can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing a polypeptide of the invention can be used to immunize an animal to produce an antibody. The antibody may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J.
  • Animals of the invention can be used to immunize animals, such as rabbits, mice, rats and the like.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • the invention also provides the use of said minimal motif peptide for identifying the presence of HPV 18, 16, 33, 52 and 58 viruses in a sample.
  • the minimal motif peptide of the present invention one skilled in the art can sensitively detect the presence or absence of a minimal motif peptide sequence in a sample by various means, and the technique employed may be a technique commonly used in the field of immunology such as PCR. method.
  • the present invention also provides the epitope minimization peptide and extended short peptide alone (such as fusion expression with GST188 protein) or by combining (constructing a recombinant multi-epitope peptide) for preparation of serological diagnosis of high-risk HPV18 and HPV16, Detection antigens for antibodies infected with 33, 52 and 58 viruses.
  • the present invention also provides an epitope marker pentapeptide amino acid sequence which can specifically discriminate between HPV16, 52, 33 and 58 high-risk HPV virus infections based on translated protein sequences after PCR amplification of the HPV-E6 gene.
  • the present invention further provides the use of the monoclonal antibody C1P5 for the preparation of a kit for detecting HPV infection, wherein the newly discovered high-risk HPV is an HPV virus of type 33, 52 or 58.
  • the invention may be further described by the following examples. The examples are not intended to limit the scope of the invention, which is set forth in the foregoing description.
  • the experimental methods in the following examples, which do not specify the specific conditions, are in accordance with the conventional conditions and the third edition of the translation of M. Sambrook and DW Russell, and Huang Peitang, "Molecular Cloning: Laboratory Manual” (Science Publishers, 2002) and [United States] E. Harlow and D.
  • Mouse anti-recombinant HPV18-E6 protein C1P5 monoclonal antibody [Banks L, Spence P, Androphy E, et al. Identification of human papillomavirus type 18 E6 polypeptide in cells derived J Gen Virol, 68 (Pt 5): 1351-1359, 1987] was purchased from American Research Products (AP) Company (Cat. No. 12-6080A). In immunoblotting experiments, the concentration of the monoclonal antibody used is in accordance with the recommended concentration in the instructions.
  • HPV58-E6 protein engineering bacteria a series of 15 peptide fusion proteins spanning the full length of HPV58-E6 protein overlapping 9 residues were obtained from Shanghai Institute of Planned Parenthood (Xu Wanxiang et al; human papillomavirus type 58)
  • the epitope of the E6 protein is the smallest motif peptide; Chinese Patent No.: ZL 2009 1 0196693.5).
  • the construction of the 8-peptide fusion protein containing the E6-2 epitope YGDTL motif can be found in the invention patent (Xu Wanxiang et al.
  • Chinese Patent No.: ZL 2009 1 0196693.5 The minimal epitope peptide of the epitope of human papillomavirus 58-E6 protein.
  • Biotechnology ampicillin (Amp), pre-stained protein molecular weight standards, horseradish peroxidase-labeled goat anti-mouse secondary antibody (IgG/HRP) and 0.2 ⁇ nitrocellulose membrane were purchased from Shanghai Biotech Biotechnology Services. Immunoblot chemiluminescence The ECL assay kit was purchased from GE.
  • the two ends are BamH I and Sal I cohesive ends, and the middle is the P2 ⁇ P6 short peptide coding DNA sequence plus the TAA stop codon positive and negative strand DNA fragment (based on HPV18 genomic sequence, GenBank accession number: NC-001357) Shanghai Jierui Biological Engineering Co., Ltd. synthesis.
  • HPV16 GenBank accession number: NC-001526
  • HPV18 GenBank accession number: NC-001357
  • HPV33 GenBank accession number: EU918766
  • HPV52 GenBank accession number: X74481
  • HPV58 type GenBank accession number: D90400
  • the epitope motif recognized by C1P5 mAb is not HPV58 E6-2 epitope
  • the E6-2 fine epitope motif YGDLE previously identified with rabbit anti-recombinant HPV58-E6 polyclonal antibody
  • the 22-residue E6/2 table identified with rabbit anti-recombinant HPV18-E6 polyclonal antibody
  • the peptide (sequence: IDFYS I EL HYSDSVYGDTL (SEQ ID NO: 3)) is also present at the end (100% identical), and the 10 residues of E6/1 epitope identified by murine anti-recombinant HPV16-E6 polyclonal antibody
  • the end of the peptide shows 1 of D ⁇ T ⁇ group difference (80% similar. See literature: Gao L, et al.
  • HPV58-E6 protein and the YGDTL-containing motif 8 peptide fusion protein sequence are SLYGDTLE (SEQ ID NO: 4).
  • the C1P5 mAb recognized the HPV58-E6 protein but was not reactive with the short peptide containing the E6-2 epitope.
  • the C1P5 mAb recognizes the HPV58/E6 protein, it induces a series of 15 polypeptides (numbering No. l to No. 23, which overlaps the full length (149aa) of the HPV58-E6 protein by overlapping 9 residues. Where ⁇ 23 is a 17-polypeptide) (fusion protein). After completion of SDS-PAGE, the nitrocellulose membrane was transferred, and the C1P5 monoclonal antibody was again used for immunoblotting.
  • the C1P5 mAb recognizes another lj L LLSKISEY HYNYfSEO ID NO: 5) and ISEY HYNYSLYGDTfSEO ID NO: 6) Two 15 polypeptides (fusion proteins) adjacent to each other with 9 residues overlapping, thus determining the single The anti-recognized epitope motif is located within the overlapping region of 9 residues of two adjacent 15-polypeptides (ISEYRHYNY (SEQ ID NO: 7)).
  • the amount of work required for short peptide biosynthesis is based on the 9-peptide motif known from the above-mentioned immunoassay, based on the published information of the HPV16, 18 and 58 E6 protein sequences. Source protein sequence alignment.
  • the C1P5 mAb recognition epitope motif was reduced to the EY(L)RHY 5 peptide, and since one residue was extended to the N-terminus or the C-terminus, two residue differences occurred.
  • the epitope motif recognized by the monoclonal antibody is often 1 to 2 residues shorter than the epitope motif recognized by the polyclonal antibody.
  • ELRHY based on the HPV18/E6 protein sequence ELRHY, four short peptides with one residue were subtracted one by one from the N-terminus (three alanine residues were added to the N-terminus, number P2-P5). ), and a P5 short peptide corresponding to the HPV16/HPV58 homologous protein sequence, and an AAA-EYRHY short peptide (P6) (SEQ ID NO: 8).
  • the GST188 coding sequence), 1 ⁇ ⁇ 4 DNA ligase and its 1.5 ⁇ M buffer were ligated overnight; the lignin was transformed into competent BL21 (DE3) host bacteria, coated on Amp-containing LB plates, and cultured at 37 ° C overnight; On the next day, the monoclonal transfer 3 ml LB medium grown on the Amp-added LB plate was used to induce expression, and the GST188-peptide expressed by pXXGST-2 was used as a control, and each expressed GST188-short peptide (P2-P6).
  • the fusion protein was confirmed by 15% SDS-PAGE analysis (about 1 kDa difference from the electrophoretic mobility of the control protein), and each recombinant clone was picked and sent for DNA sequencing verification.
  • the clones with the correct sequencing results of the inserts were inoculated into 3 ml of LB culture medium of Amp, and cultured overnight at 30 ° C with shaking.
  • the fresh LB-containing LB medium was transferred to the LB medium at a ratio of 1/50, and cultured at 30 ° C with shaking. 2 ⁇ 3 h After the concentration of the cells reached 0.6 ⁇ 0.8 OD, the temperature was increased to 42 ° C for 4 h, and the cells were collected by centrifugation, and the sample lysate was boiled for 5 min.
  • the induced total bacterial protein samples were simultaneously subjected to 15% SDS-PAGE. After electrophoresis, one gel was stained with Coomassie brilliant blue, and two gels were transferred to nitrocellulose membrane (100 mA) for 2 h, using Li Chunhong. The blot was transferred to the blotting membrane for 2 min, and the needle was punched with a needle at the band of the short peptide fusion protein of interest, and the Ponceau red stain was washed away with water.
  • the blotting membrane was washed repeatedly four times with PBS buffer, blocked with 5% skim milk powder overnight, washed four times with PBS buffer, and added with 1 ⁇ M anti-C1P5 monoclonal antibody in 8 to 10 ml reaction solution, and reacted at room temperature for 2 h, PBS. After washing with buffer, 5 ⁇ l of secondary anti-mouse anti-mouse IgG/HRP (l: 2000 dilution) was added, and the reaction was carried out for 1 h at room temperature. After washing with PBS buffer, ECL chemiluminescence was performed, and P5 and P6 were confirmed by immunoblotting.
  • the short peptide (fusion protein) confirms that the minimal motif recognized by C1P5 mAb is ELRHY and is cross-reactive with the HPV16/HPV58 homologous protein conserved EYRHY 5 peptide.
  • Candidate B cell epitopes of HPV multi-epitope vaccines can also be used as candidate B cell epitopes for the detection of antigens by multi-epitope peptides for the development of high-risk HPV virus-infected serum antibodies by chemical synthesis or fusion expression or alone or in combination.
  • C1P5 mAb cross-reactive pentapeptide sequence is described, which can be used alone or in combination with HPV58 E6-2 highly conserved epitope motifs (YG-D/X) present in high-risk HPV-E6 proteins.
  • -TL wherein the X residue is T in HPV16, ⁇ in 33, ⁇ in 52, and D) in 58, and is used to PCR-amplify HPV virus E6 protein gene or fragment to determine infection with high-risk HPV type (16) Reference peptide sequences of 18, 33, 52 and 58).

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Abstract

提供了一种人乳头瘤病毒（HPV）E6蛋白表位最小基序，其存在于包括HPV16、18、33、52或58型的多种HPV亚型的E6蛋白中，可诱发同源蛋白间的交叉反应性抗体。

Description

人乳头瘤病毒 E6蛋白可诱发同源蛋白间
交叉反应性抗体的精细表位肽
技术领域
本发明属于免疫学和生物医药技术领域， 具体涉及一种人乳头瘤病毒 E6 蛋 白中能被单克隆抗体 C 1P5识别的最小基序肽及其应用。 背景技术
宫颈癌是目前世界上导致女性死亡的第二大癌症， 其威胁仅次于乳腺癌。 全 球每年约有 50万人被诊断为宫颈癌， 约有 30万人死于此癌症， 而中国的每年发 病率则估计在 13.5万， 死亡人数 5万。 上世纪 80年代初就确定了人乳头状瘤病 毒 (human papillomavirus, HPV)与宫颈癌的相关性。最新资料表明 99.8%子宫颈癌 活检标本中可检出 HPV。 因此， 关注妇女生殖健康， 各国科研人员长期以来一直 就 HPV的诊断、 预防和治疗的方法途径展开研究。 令人兴奋的是， 2006和 2007 年首先已由美国默克 (Merck)和英国葛兰素史克 (GSK)制药公司研制成功二种预防 性四价和二价 HPV疫苗。
但是， 就 HPV预防和治疗而言， 目前这方面需要人类攻克的课题依然很多。 例如， 现已清楚感染女性生殖道等上皮组织的 HPV型有 120种之多， 其中可经持 续感染导致子宫颈癌的高危型 HPV则存在 20种左右 [de Villiers EM, Fauquet C， Broker T , et al. Classification of papillomaviruses. Virol 2004 , 324： 17-27； Parkin DM. The global health burden of infection- associated cancers in the year 2002. Int J Cancer, 1 18： 3030-3044 , 2006； de Sanjose S , Quint WG, Alemany L, et al. Human papillomavirus genotype attribution in invasive cervical cancer： a retrospective cross-sectional worldwide study. Lancet Oncol 2010 , 1 1(1 1)： 1048-1056] , 而目前已 被许多国家批准临床应用的以上 Gardasil和 Cervarix预防性 HPV疫苗主要是针对 流行率最广的 HPV16和 18型的，虽然它们对其他一些高危型 HPV也显示出一定 程度的保护作用 [Wheeler CM， Castellsague X, Garland SM, et al. Cross-protective efficacy of HPV 16/ 18 ASO4-adjuvanted vaccine against cervical infection and precancer caused by non-vaccine oncogenic HPV types: 4-year end-of-study analysis of the randomized, double-blind PATRICIA trial. Lancet Oncol, 13(1) : 100-1 10， 2012]。 因此， 下一个已受到普遍关注的课题无疑是研发 "通用" HPV疫苗， 这对 中国和亚洲妇女尤为重要， 因为在这一地区妇女感染高危 HPV型的流行率排序， 在 HPV16之后主要是 HPV58和 HPV52 , 而非通常排第二的 HPV18 [Huang S， Afonina I， Miller BA， Beckmann AM. Human papillomavirus type 52 and 58 are prevalent in cervical cancers from Chinese woman. Int J Cancer, 70(4)： 408-41 1， 1997 等 1。
此外，由于默克和葛兰素史克制药公司 HPV四价和二价疫苗生产工艺复杂和 综合成本高， 其疫苗价格显然不适合在发展中国家推广应用， 因而研制易制备储 存价格低等突出优点的重组多表位肽疫苗， 也是一个当前疫苗发展的重要方向。 虽然目前的 HPV治疗性多肽疫苗多选择 HPV16或 18型 E6和 E7蛋白上的细胞 毒性 T细胞表位 (TCL/ TcCE)肽， 但也有研制靶向早期致癌性 E6和 E7蛋白， 同 时诱发 CD4+、 CD8+和抗体应答的治疗性 HPV融合蛋白疫苗报道，提示也可研制 组合它们 B-和 T-细胞表位的预防性 HPV多表位肽疫苗， 即通过产生抗体的体液 应答， 以及在癌前病变或宫颈恶性病变前排除它们对肿瘤特异性免疫应答而达到 完全或部分清除 HPV效果。
另外， 在 HPV感染临床门诊方面， 建立介于细胞学观察和 DNA诊断之间简 便易行灵敏准确的血清学检测方法， 也始终是一项重要的课题。 即， 作为这一方 法学的核心内容或技术，就是鉴定 HPV感染者血清中存在的抗体及其线性抗原表 位 [Dillner J. Mapping of linear epitopes of human papillomavirus type 16： the El， E2， E4， E5， E6 and E7 open reading frames. Int J Cancer, 46： 703-711， 1990等]， 然后以合成表位肽为 ELISA包被抗原建立 ELISA检测方法。 但现实是， 至今未 见血清学检测方法或 Kit在临床检验中的实际应用， 原因就在于难以排除 ELISA 检测中常见但医患双方都不希望出现的高比例假阳性结果。 可以想见， 假阳性结 果由是以下三个因素合并形成， 即： 1)一般患者体内应答 HPV感染的抗体滴度通 常不高， 因而血清样品稀释度不可能高 (常见用 1 :2- 1 : 10血清稀释度)； 2)因抵御病 毒等微生物感染和自身免疫等因素， 人体内往往存在成千上万难以计数的抗不明 抗原抗体； 3)以使用检测单一抗体的 18-20 个组成单表位肽作为检测抗原为例 [Dillner J， Dillner L, Cheng HM. Synthetic peptides in human papillomavirus 1， 5， 6， 8， 1 1， 16， 18， 31， 33 and 56， useful in immunoassay for diagnostic purposes. United States Patent Number: 5932412, 1999]， 已知线性 B细胞表位通常由 3-8 残基组成， 因此大于 8个残基的表位肽上显然潜在能被体内不明抗原抗体识别的 N个表位， 就存在低血清稀释度情况下被体内某一或若干抗体识别的几率。 基于 以上认知， 今后研制高特异性高灵敏度合成肽检测用抗原， 需要创新突破的方向 应该是： 1)检测抗原最好使用表位最小基序或扩展八肽， 以使与体内抗不明抗原 抗体的交叉反应性降到最低， 从而提高检测的特异性； 2)构建基于最小基序的多 表位肽抗原， 以测定靶蛋白表面多个表位抗体， 通过富集它们的低滴度抗体以及 偶联二抗的放大效应扩大待测血清样品的稀释度， 提高目标抗体的检测灵敏度。 而体内的同样低滴度非目标抗体， 则随着血清稀释度的扩大而下降至检测灵敏度 以下， 从而减少可能的交叉反应性， 避免假阳性结果。 很显然， 以上检测抗原 Kit 研制目标的实现有赖于靶抗原更多线性表位及其最小基序的鉴定。
基于以上 HPV 疫苗和诊断试剂研发的实际需求背景， 本发明人先前已完成 HPV58型 E6蛋白的全部四个线性表位及其精细基序鉴定 [徐万祥等； 人乳头瘤病 毒 58型 E6蛋白的抗原表位最小基序肽； 中国专利申请号： 200910196693.5]， 并 发现了其中 HPV58型 E6-2表位 YGDTL84—88基序在高危型 HPV同源蛋白之间具 有高度保守性 (图 1)， 且与被鉴定的 HPV18型显示为 22肽的 E6/2表位 C端末的 5个残基序歹 [J完全相同 [Bleul C Muller M, Frank R，et al. human Papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients. J Clin Microbiol, 29(8)： 1579- 1588， 1991 ]。
众所周知， 一个抗原表位代表一种抗体， 一个抗体对应一个表位， 特别是表 位和抗体在抗病毒抗肿瘤药物 (疫苗）以及诊断试剂等方面的可能应用， 因而无容 置疑表位的鉴定或制备都具有发明和制备的知识产权。 当然， 作为这方面知识产 权的形成还在于鉴定或制备技术的难度。例如， Banks L等用重组 HPV18型 E6蛋 白制备了包括 C 1P5 在内六株单抗 [Banks L， Spence P， Androphy E， et al. Identification of human papillomavirus type 18 E6 polypeptide in cells derived from human cervical carcinomas. J Gen Virol, 68 ( Pt 5)： 1351 -1359 , 1987]， 但都未进 行各株单抗识别表位基序的鉴定， 从而未能发现 C1P5单抗除 HPV16-E6蛋白以 外与其他更多高危型 HPV 同源蛋白之间的交叉性反应 (因此失去其更多的应用范 围）， 以及确定另外五株命名为 D2A6、 C 1N1、 C1X B 1B3和 B 1A2单抗识别的 表位。 关于后者其他 5个单抗识别表位基序的鉴定也很重要， 因为尽管五株单抗 亚型不同， 也存在 2株或更多不同亚型单抗识别同一个表位基序的可能性。例如， 在另一项研究中，两株抗 HPV18-E6不同亚型 (G3和 G1)单抗都同时识别 PTRR7_1Q 四个残基基序 [Bleul C， Muller M， Frank R， et al. human Papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients. J Clin Microbiol, 29(8)： 1579- 1588， 1991 ]。 但是受表位鉴定方法学的限 制， 以往这方面研究报道并不多。 原因在于即便相对单抗而言， 应用包括化学合 成肽法在内的表位鉴定技术操作非易事。 因此，应用改良生物合成肽法来鉴定新的单抗识别表位基序 HPV精细表位肽 在本领域中具有重要意义。 发明内容
本发明的目的在于提供一种人乳头瘤病毒 E6蛋白中能被单克隆抗体 C1P5识 别的最小基序及其应用。
在本发明的第一方面， 提供一种分离的多肽， 所述的多肽由式 (I)所示的氨基 酸序列组成；
EX HY (1)；
其中， X选自氨基酸 L或 Y;
并且， 所述的多肽来源于人乳头瘤病毒 (HPV)的 E6蛋白。
在一个优选例中， 所述的多肽具有 SEQ ID NO: 1或 SEQ ID NO: 2所示的氨 基酸序列。
在另一优选例中，所述的多肽来源于人乳头瘤病毒 18型 (X为 L)、 16型 (X为 Y)、 52型 (X为 Υ)、 33型 (X为 Υ)或 58型 (X为 Υ)的 Ε6蛋白。 在本发明的另一方面， 提供前面所述的多肽的用途， 用于制备抗人乳头瘤病 毒 Ε6蛋白的免疫原。
在一个优选例中， 所述的人乳头瘤病毒是 18、 16、 52、 33或 58型。 在本发明的另一方面， 提供前面所述的多肽的用途， 用于制备可特异性结合 人乳头瘤病毒的 Ε6蛋白的抗体； 较佳地， 所述的人乳头瘤病毒是 18、 16、 52、 33或 58型。 在本发明的另一方面， 提供所述的多肽的用途， 用于制备单独或组合构建血 清学诊断 HPV18感染的多表位肽检测抗原。 在本发明的另一方面， 提供前面所述的多肽的用途， 用作在 PCR扩增 Ε6基 因后依据它们的编码氨基酸序列用作判别 HPV18、 16、 52、 33或 58型感染的表 位标志物或参照物。 在本发明的另一方面， 提供单克隆抗体 C1P5 的用途， 用于制备检测人乳头 瘤病毒的试剂盒， 其中， 所述的人乳头瘤病毒是 58型、 33型或 52型的病毒。 在本发明的另一方面， 提供一种检测人乳头瘤病毒的试剂盒， 所述的人乳头 瘤病毒是 58型、 33型或 52型的病毒， 所述试剂盒中包括单克隆抗体 C1P5。 本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易 见的。 附图说明
图 1、HPV58-E6蛋白 E6-2表位基序在高危型 HPV同源蛋白中的保守性分析。 方框中的 HPH58型 E6-2基序 YGDTL, 与 HPV18-E6蛋白中相应序列具 100%保 守性。 其他高危型 HPV同源蛋白序列与之比对， 存在同一位置的 1个残基差异， 但它们与截短 GST188融合表达的八肽都能被抗重组 HPV58-E6蛋白多抗识别。 表位基序相似率依据方框内 5个残基计算。 图 2、 HPV18-E6单抗 C1P5可能识别表位基序的 HPV同源蛋白序列比较确 定。 图中高危型 HPV同源蛋白相似序列比对依据 C1P5单抗识别 HPV58-E6蛋白 9个残基免疫印迹结果。 方框内以 HPV18序列为基准， 仅 1个残基差异的 5个残 基序列代表 C1P5单抗可能识别的表位最大基序。 图 3、 短肽融合表达的 SDS-PAGE分析 (A)、用 C1P5单抗的印迹鉴定 (B)和反 应性最小基序肽的列表图示 (C)。其中， Lane 1为 GST188蛋白阴性对照， Lanes 2〜 6， GST188-P2〜P6融合蛋白 (A)。 印迹膜上箭头指示反应性短肽融合蛋白 (B)。 图 C中各短肽前添加 3个 AAA残基， 一是以方便合成短肽编码 DNA片段的重组克 隆， 二是以排除 GST188载体蛋白 C端末 BamH I限制酶位点产生两个残基对表 位鉴定可能的干扰。 具体实施方式
本发明人经过广泛地研究， 首次用市售 C1P5单抗定位到了 HPVE6蛋白序列 上一特定的精细表位基序， 所述的抗原表位在多种 HPV亚型 (包括： 16、 18、 33、 52或 58型)病毒的 E6蛋白中高保守性地存在。 因此， 利用含有该表位的多肽 (本 发明中还称为短肽或基序肽)，可以制备抗 HPV E6蛋白的免疫原 (与其他表位组合 构建多表位肽免疫原)， 或制备特异性抗 HPV E6蛋白的抗体， 所获得的免疫原或 抗体可用于多种 HPV亚型的防治和诊断。 术语
如本文所用， "分离的"是指物质从其原始环境中分离出来 (如果是天然的物 质， 原始环境即是天然环境)。 如活体细胞内的天然状态下的多聚核苷酸和多肽是 没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质 中分开， 则为分离纯化的。
如本文所用， "E6蛋白"是指存在于 HPV的一种蛋白， 本发明中， HPV16、 HPV18、 HPV33 , HPV52和 HPV58型的 E6蛋白编码基因序列和蛋白质一级结构 序列分别依据它们的标准株， 它们的 GenBank登录号依次分别为： NC— 001526、 NC— 001357、 EU918766, X74481禾 Π D90400。
如本文所用， 所述的 "含有" ， "具有" 或 "包括" 包括了 "包含" 、 "主 要由 ......构成" 、 "基本上由 ......构成" 、 和 "由 ......构成" ； "主要由 ......构 成" 、 "基本上由 ......构成" 和 "由 ......构成" 属于 "含有" 、 "具有 " 或 "包 括" 的下位概念。
如本文所用， 抗体的 "特异性" 是指抗体能结合于特定亚型的 HPV E6蛋白 或片段。 特别指那些能与特定亚型的 HPV E6蛋白结合的抗体。 抗原表位及其用途
本发明人在研究过程中， 分析了一株商品化的 HPV18/E6单抗 (C1P5单抗)， 以确定它是否识别 HPV58/E6-2表位，进而确定此鼠单抗 C1P5识别 HPV18型 E6/2 表位的精细基序。 分析过程中， 本发明人出乎意料地发现， C1P5 单抗能与重组 HPV58型 E6蛋白产生免疫印迹反应， 但并不识别含 YGDTL基序八肽。 继而， 采用独特策略开展相关表位鉴定研究， 最终形成本发明的技术方案。
本发明人的预实验结果表明， HPV18-E6蛋白 C1P5单抗可与重组 HPV58-E6 蛋白发生免疫印迹交叉反应， 但不识别在同源蛋白具高度保守性的含 HPV58 型 E6-2最小基序 YGDTL (在同源蛋白中具高度保守性， 图 1)八肽融合蛋白。 于是， 用相互重叠的系列 HPV58-E6蛋白 15聚肽融合蛋白进行免疫印迹鉴定，结果表明 C1P5单抗识别 LRLLSKISEYRHYNY禾口 ISEYRHYNY SLYGDT两个相互重叠 9 个残基的 15聚肽，也就是说，该单抗识别基序落在 9个残基的重叠区 (ISEYRHYNY) 内。 再进一步比对 HPV16、 18和 58型 E6蛋白序列， 如图 2所示， C1P5单抗识 别的最小基序又进一步被缩减至相互间仅 1个残基差异的 ELRHY五肽基序， 因 为若再向左或向左右延伸 1个残基形成六肽基序， 那么三个蛋白序列都显示 2个 残基差异， 就不可能被 C1P5单抗识别。 最后， 为了鉴定 C1P5单抗可识别的表位 最小基序， 如图 3所示， 重新构建表达了 HPV18型 P2-P5四个和 HPV16/18型 P6 一个短肽融合蛋白（图 3A)， 在免疫印迹试验中仅 P5和 P6短肽产生印反应性条带 (图 3B)，因此确定 C1P5单抗识别 HPV18-E6蛋白的表位最小基序为 ELRHY五肽， 而且是 HPV18型特异的表位基序 (图 2)， 并证实了与 HPV16/58型发生交叉反应 的一个保守性 EYRHY五肽序列（图 3B和 3C：)。 另外， 通过同源蛋白序列比较， 除了高危型 HPV16和 58型， 发现 HPV18型 C1P5单抗还可与高危型 HPV33和 HPV52型 E6蛋白发生交叉反应，其序列中也存在与 HPV16/58反应性五肽完全相 同的保守性序列（图 2)。
基于本发明人的新发现， 提供了一个线性表位的最小基序肽， 具有 EXRHY 所示的氨基酸序列； 其中， X选自氨基酸 L或 Y; 并且， 所述的多肽来源于五种 高危型 HPV的 E6蛋白。 作为本发明的优选方式， 所述的多肽具有 SEQ ID NO: 1 (ELRHY)或 SEQ ID NO: 2(EYRHY)所示的氨基酸序列。
一旦获得了所述多肽的序列， 就可以用重组法来大批量地获得该多肽融合蛋 白。 这通常是将其克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主 细胞中分离得到有关序列的融合蛋白。 此外， 对比本发明的短肽而言， 优选的是 采用人工合成（如通过多肽合成仪合成）的方法来合成有关序列， 人工合成的方法 可简便且快速地得到所需要的多肽。
本发明同时提供所鉴定的表位最小基序肽和其扩展肽在制备 "通用" 预防性 /治疗性 HPV重组多表位肽疫苗中的应用。
本发明所述的最小基序肽可作为 E6 蛋白的一种有效的抗原性的片段， 用于 制备特异性结合 E6 蛋白的通用型抗体， 包括单克隆抗体或多克隆抗体。 本发明 所述的最小基序肽还可与其它肽或免疫学上可接受的载体 (最好其本身没有免疫 原性)连接， 以用于作为免疫原制备抗体。
抗体可以通过本领域内技术人员已知的各种技术进行制备。 例如， 纯化的本 发明的多肽 (或与其它肽或免疫学上可棘手的载体连接后)可被施用于动物以诱导 多克隆抗体的产生。 与之相似的， 表达本发明的多肽的细胞可用来免疫动物来生 产抗体。 所述的抗体也可以是单克隆抗体。 此类单克隆抗体可以利用杂交瘤技术 来制备（见 Kohler等人， Nature 256； 495， 1975； Kohler等人， Eur. J. Immunol. 6： 511， 1976； Kohler等人， Eur. J. Immunol. 6： 292, 1976； Hammerling等人， In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, Ν·Υ·， 1981)。 多克隆抗 体的生产可用本发明的多肽免疫动物， 如家兔， 小鼠， 大鼠等。 多种佐剂可用于 增强免疫反应， 包括但不限于弗氏佐剂等。
本发明还提供了所述的最小基序肽的用途， 用于鉴定样品中是否存在 HPV18、 16、 33、 52和 58型的病毒。 在获得了本发明的最小基序肽后， 本领域 人员可通过多种途径来灵敏地检测样品中最小基序肽序列的存在与否， 采用的技 术可以是免疫学领域中常用的技术如 PCR方法。
本发明也提供所述的表位最小基序肽和扩展短肽单独 (如与 GST188蛋白融合 表达)或通过组合 (构建重组多表位肽)， 制备用于血清学诊断高危型 HPV18 以及 HPV16, 33、 52和 58病毒感染抗体的检测抗原。
本发明还提供了可用于 PCR扩增 HPV-E6基因后， 依据翻译蛋白质序列特异 性地判别 HPV16、 52、 33和 58型四个高危型 HPV病毒感染的表位标志物五肽氨 基酸序列。 单克隆抗体 C1P5的新用途
已有技术中， 仅发现 C1P5单抗能够识别 HPV18-E6蛋白和 HPV16-E6蛋白， 而并不清楚 C1P5单抗是否针对其它病毒亚型的 E6蛋白也具有识别作用。而本发 明意外地开拓了单克隆抗体 C1P5的新用途。
因此， 本发明进一步还提供了单克隆抗体 C1P5的用途， 用于制备检测 HPV 感染的试剂盒， 其中， 所述的新发现高危型 HPV是 33、 52或 58型的 HPV病毒。 本发明可进一步通过下列实例描述。 列举实例并不意味限制本发明所涉及的 范围， 该范围在之前的描述中已被充分陈述阐明。 下列实施例中未注明具体条件 的实验方法， 均按照常规条件以及 [美] J. 萨姆布鲁克和 D.W. 拉塞尔编著、 黄培 堂等翻译的第三版 "分子克隆： 实验室手册"（科学出版社， 2002)和 [美] E. 哈洛 和 D. 莱恩编著沈关心等翻译的 "抗体技术实验指南" （科学出版社， 2002)中所 述的步骤， 或按照生产销售商建议的条件进行。 实施例 1、 用重组 HPV18-E6蛋白 C1P5株单抗的精细表位鉴定
一、 材料
1. 鼠抗重组 HPV18-E6蛋白 C1P5单抗 [Banks L， Spence P, Androphy E, et al. Identification of human papillomavirus type 18 E6 polypeptide in cells derived from human cervical carcinomas. J Gen Virol, 68 ( Pt 5)： 1351-1359， 1987] 购自 American Research Products (A P) 公司 （商品号为 12-6080A)。 在免疫印迹实验 中， 其单抗使用浓度参照说明书中建议浓度。
2. HPV58-E6蛋白工程菌、 跨越 HPV58-E6蛋白全长序列相互重叠 9个残基 的系列 15肽融合蛋白重组克隆获自上海市计划生育科学研究所 （徐万祥等； 人乳 头瘤病毒 58 型 E6 蛋白的抗原表位最小基序肽； 中国专利号： ZL 2009 1 0196693.5)。 含 E6-2表位 YGDTL基序的 8肽融合蛋白的构建见发明专利 (徐万祥 等. 人乳头瘤病毒 58-E6 蛋白的抗原表位最小基序肽. 中国专利号： ZL 2009 1 0196693.5)
3. 大肠杆菌 BL21(DE3)宿主菌和专门用于抗原表位扫描作图的短肽生物合 成的 pXXGST-Ι和仅表达作为阴性对照截断 GST188蛋白的 pXXGST-2质粒， 获 自上海市计划生育科学研究所 [Xu WX, et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy. J eprod Immunol, 81： 9-16， 2009]。
4. 限制性内切酶 BamH I、 Sal I 和 T4 DNA 连接酶购自日本 TaKaRa
Biotechnology 公司， 氨苄青霉素 (Amp)、 预染蛋白分子量标准、 辣根过氧化酶标 记的羊抗鼠二抗 (IgG /HRP)和 0.2 μιη硝酸纤维素膜购自上海生工生物技术服务公 司。 免疫印迹化学发光 ECL检测试剂盒购自 GE公司。
5. 两端分别为 BamH I和 Sal I粘性末端， 中间为 P2〜P6短肽编码 DNA序 列加 TAA终止密码子的正负链 DNA片段 (依据 HPV18基因组序列， GenBank登 录号： NC— 001357)由上海捷瑞生物工程有限公司合成。
6. 高危型 HPV16 (GenBank登录号： NC— 001526)、 HPV18(GenBank登录号： NC— 001357)、 HPV33(GenBank 登录号： EU918766)、 HPV52(GenBank 登录号： X74481)和 HPV58型 (GenBank登录号： D90400)E6蛋白序列信息均出自 GenBank 数据库。 二、 用 C1P5单抗的 HPV18/E6蛋白精细表位鉴定
1、 C1P5单抗识别的表位基序并非 HPV58型 E6-2表位
如图 1 所示， 之前用兔抗重组 HPV58-E6 多抗鉴定的 E6-2 精细表位基序 YGDLE,在用兔抗重组 HPV18-E6多抗鉴定的长 22个残基的 E6/2表位肽 (序列为： IDFYS I EL HYSDSVYGDTL(SEQ ID NO: 3))末尾也存在（100%相同），在用鼠抗 重组 HPV16-E6多抗鉴定的长 10个残基的 E6/1表位肽末尾则显示 D→T的 1个 歹戋基差异 (80%相似。见文献： Gao L， et al. Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector. J Gen Virol, 75： 157-164， 1994), 但其 8 肽序列也能被 HPV58-E6 多抗识别。 因此最初推断同时能识别 HPV16-E6 的 HPV18-E6单抗 (C1P5株)或许也能识别 HPV58/E6-2表位。 因此， 本发明人利用 C1P5单抗以证实这一推断，并通过其识别最小基序鉴定探讨兔多抗和鼠单抗识别 表位基序长短是否存在变化。于是， 诱导表达 HPV58-E6蛋白和含 YGDTL基序 8 肽融合蛋白， 完成 SDS-PAGE后转硝酸纤维素膜， 用 C1P5单抗进行免疫印迹鉴 定。
HPV58-E6蛋白和含 YGDTL基序 8肽融合蛋白序列为 SLYGDTLE(SEQ ID NO: 4)。
免疫印迹鉴定结果， C1P5单抗能识别 HPV58-E6蛋白， 但与含 E6-2表位基 序短肽无反应性。
2、 C1P5单抗识别位点筛选
既然 C1P5单抗可识别 HPV58/E6蛋白，于是诱导表达跨越 HPV58-E6蛋白全 长 (149aa)序列相互重叠 9个残基的系列 15聚肽 (共 23个， 编号 No. l 〜 No. 23， 其中 Νο·23为 17聚肽） （融合蛋白）。 完成 SDS-PAGE后转硝酸纤维素膜， 再次用 C1P5单抗进行免疫印迹鉴定。
结果， C1P5 单抗同时识另 lj L LLSKISEY HYNYfSEO ID NO: 5)和 ISEY HYNYSLYGDTfSEO ID NO: 6) 两个相邻互有 9个残基重叠的 15聚肽 (融合 蛋白）， 因此可确定该单抗识别的表位基序位于两个相邻 15聚肽的 9个残基的重 叠区（ISEYRHYNY(SEQ ID NO: 7))内。
3、 序列分析以縮减 C1P5单抗识别表位基序
为了简化鉴别 C1P5单抗识别表位最小基序而需进行短肽生物合成的工作量， 以上述免疫鉴定获知的 9肽基序为基准， 依据 HPV16、 18和 58型 E6蛋白序列公 开信息进行同源蛋白序列比对。
结果如图 2所示， C1P5单抗识别表位基序被缩减至 EY(L)RHY 5肽， 因为向 其 N端或 C端延伸 1个残基， 都将出现两个残基差异。
4、 免疫印迹验证 5肽最小基序
因为单抗识别的表位基序往往比多抗识别的表位基序短 1〜2个残基，因此如 图 3C所示， 以 HPV18/E6蛋白序列 ELRHY为基准， 设计从其 N端起逐一减去 1 个残基的 4 个短肽 (N 端分别增加 3 个丙氨酸残基， 编号 P2-P5), 以及 1 个与 HPV16/HPV58同源蛋白序列对应的 P5短肽，和一个 AAA-EYRHY短肽 (P6) (SEQ ID NO: 8)。 基于所设计的短肽， 分别依据如上所述公开 HPV基因组信息设计它们 的编码 DNA 正负链片段 (正链 5'-端加 5'-gatcc， 3'-端加 taag-3' ; 负链 5'-端加 5，-tcgactta， 3，-端加 g-3，）， 外送 DNA化学合成。
将 1或 2个 OD的互补正负链片段用 ddH2O溶解成 20 μιηοΐ/μΐ储存液 (根据 DNA合成报告单数据）； 各取 ΙΟ μΙ储存液和 20 ddH2O于 1.5 ml Eppendorf管中， 94°C水浴加热 5 min， 自然降至室温后， 在 15 μΐ反应体积中吸入 2 μΐ退火片段、 1 μΐ 约 200 ng/μΐ经 BamH I和 Sal I双酶切的 pXXGST-1质粒（自带 GST188编码 序列）、 1 μΐ Τ4 DNA连接酶和其 1.5 μΐ缓冲液， 连接过夜； 连接液转化感受态 BL21(DE3)宿主菌， 涂布含 Amp的 LB平板上， 于 37°C培养过夜； 第二天挑取在 添加 Amp 的 LB 平板上长出的单克隆转接 3 ml LB 培养液诱导表达， 以由 pXXGST-2表达的 GST188蛋白为对照， 各表达的 GST188-短肽 (P2-P6)融合蛋白 经 15%SDS-PAGE分析确认 (与对照蛋白电泳迁移率相差约 1个 kDa)，挑取各重组 克隆外送进行 DNA测序验证。
将插入片段测序结果正确的克隆接种加入 Amp的 3 ml LB 培养液中，于 30°C 振荡培养过夜， 翌日晨按 1/50比例转接新鲜含 Amp的 LB培养液中， 30°C振荡培 养 2 〜 3 h 至菌体浓度达到 0.6 〜 0.8 OD后， 调高温度至 42°C热诱导 4 h， 离 心收集菌体， 加上样裂解液煮沸 5 min备用。
将诱导的细菌总蛋白样品同时走 15%SDS-PAGE， 电泳结束后， 一块凝胶用 考马斯亮蓝染色， 二块凝胶进行硝酸纤维素膜电 (100 mA)转移 2 h， 用丽春红染色 转印迹膜 2 min，在目的短肽融合蛋白条带处用针头打孔标记， 用水冲洗掉丽春红 染色。
印迹膜用 PBS缓冲液反复洗涤四次， 用 5%脱脂奶粉封闭过夜， PBS缓冲液 洗涤四次， 分别在 8 〜 10 ml 反应液中加入 1 μΐ—抗 C1P5单抗， 室温反应 2 h， PBS缓冲液洗涤后加入 5 μ1二抗羊抗鼠 IgG /HRP(l : 2000稀释）， 室温反应 1 h， 用 PBS缓冲液洗涤后进行 ECL化学发光显色， 依据免疫印迹结果确认 P5和 P6 为反应性短肽（融合蛋白）， 确认 C1P5 单抗识别的最小基序为 ELRHY , 且与 HPV16/HPV58同源蛋白保守性 EYRHY 5肽存在交叉反应性。
5、 分析 C1P5单抗与其它 HPV中同源蛋白的交叉反应性 最后以及 GenBank信息库中高低危型 HPV同源蛋白序列公开信息， 经过序 列比对（图 2)， 确定 C1P5单抗与高危型 HPV33和 HPV52同源蛋白也存在交叉反 应性（因为它们的 E6蛋白也存在 EYRHY序列）。 本发明描述了 HPV18-E6蛋白上一个可被 C1P5单抗识别的线性抗原表位最 小基序肽 （注： 因为国外早已鉴定公布了含这一基序的 22 肽， 因此本专利扩展 20肽不再具有新颖性，而作为封闭式表位肽专利申请，其新颖性则是显而易见的， 故删除之。 下同） ， 它们既可以单独和 /或被组合用作研制"通用"预防性 /治疗性 HPV多表位疫苗的候选 B细胞表位，也能通过化学合成或融合表达或单独或被组 合用作研制高危型 HPV病毒感染血清抗体的多表位肽检测抗原的候选 B细胞表 位。 此外， 也描述了一个 C1P5 单抗交叉反应性五肽序列， 它可单独或结合在高 危型 HPV-E6蛋白中都存在的 HPV58型 E6-2高保守性表位基序 (YG-D/X-TL, 其 中 X残基在 HPV16中为 T， 33中为 Η， 52中为 Κ， 和 58中为 D)， 用作 PCR扩 增 HPV病毒 E6蛋白基因或片段后判定感染高危 HPV型 (16， 18， 33 , 52和 58) 的参照肽序列。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 需要说明的是， 有一点对于相关领域研究技术人员来说 是显而易见的， 即在不脱离本发明的精神和范围的情况下， 可对本发明的最小表 位肽和其融合蛋白作各种变化改动。 因此， 所附权利要求覆盖了所有这些在本发 明范围内的变动。

Claims

权 利 要 求
1. 一种分离的多肽，其特征在于，所述的多肽由式 (I)所示的氨基酸序列组成;
EX HY (1)；
其中， X选自氨基酸 L或 Y;
并且， 所述的多肽来源于人乳头瘤病毒的 E6蛋白。
2. 如权利要求 1所述的多肽， 其特征在于， 所述的多肽具有 SEQ ID NO: 1 或 SEQ ID NO: 2所示的氨基酸序列。
3. 如权利要求 1所述的多肽， 其特征在于， 所述的多肽来源于人乳头瘤病毒 18型、 16型、 52型、 33型或 58型的 E6蛋白。
4. 权利要求 1-3任一项所述的多肽的用途， 用于制备抗人乳头瘤病毒 E6蛋 白的免疫原。
5. 如权利要求 4所述的用途， 其特征在于， 所述的人乳头瘤病毒是 18、 16、 52、 33或 58型。
6. 权利要求 1-3任一项所述的多肽的用途， 用于制备可特异性结合人乳头瘤 病毒的 E6 蛋白的抗体； 较佳地， 所述的人乳头瘤病毒是 18、 16、 52、 33 或 58 型。
7. 权利要求 1-3任一项所述的多肽的用途， 用于制备单独或组合构建血清学 诊断 HPV18感染的多表位肽检测抗原。
8. 权利要求 1-3任一项所述的多肽的用途， 用作在 PCR扩增 E6基因后依据 它们的编码氨基酸序列用作判别 HPV18、 16、 52、 33或 58型感染的表位标志物 或参照物。
9. 单克隆抗体 C1P5的用途， 用于制备检测人乳头瘤病毒的试剂盒， 其中， 所述的人乳头瘤病毒是 58型、 33型或 52型的病毒。
10. 一种检测人乳头瘤病毒的试剂盒， 所述的人乳头瘤病毒是 58型、 33型 或 52型的病毒， 其特征在于， 所述试剂盒中包括单克隆抗体 C 1P5。
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