WO2017049608A1 - 人乳头瘤病毒的表位肽及其应用 - Google Patents

人乳头瘤病毒的表位肽及其应用 Download PDF

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WO2017049608A1
WO2017049608A1 PCT/CN2015/090776 CN2015090776W WO2017049608A1 WO 2017049608 A1 WO2017049608 A1 WO 2017049608A1 CN 2015090776 W CN2015090776 W CN 2015090776W WO 2017049608 A1 WO2017049608 A1 WO 2017049608A1
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peptide
short
formula
hpv
amino acids
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French (fr)
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刘日廷
徐万祥
唐海平
张富春
连文博
詹建民
李轶杰
张爱莲
王健
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刘日廷
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    • 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
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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

Definitions

  • the present invention belongs to the field of bioengineering and immunology, and in particular, relates to human papillomavirus (HPV) type 16 E6 and E7 oncoproteins which are most closely related to cervical cancer in women, and can induce all fine linear B cell epitope peptides of antibodies. And its applications.
  • HPV human papillomavirus
  • BCE B cell epitope
  • Monoclonal or polyclonal antibodies monoclonal or polyclonal
  • mature conventional chemical synthetic peptide methods, phage display library methods, different long and short peptide chip methods, and computer software are used to predict the identification of BCE peptides on target proteins of known amino acid sequences.
  • HPV Human papillomavirus
  • HPV16 Human papillomavirus
  • the present invention provides an epitope peptide of human papillomavirus and uses thereof.
  • an isolated peptide collection consisting of a peptide or polypeptide selected from the group consisting of at least two selected from the short peptides of Formula I or derived polypeptides thereof, wherein
  • X is a core fragment of 4-8 amino acids; preferably 4, 5, 6, 7, or 8 amino acids;
  • X1, X2 are none, 1, 2 or 3 amino acids, and the total number of X1 and X2 amino acids is ⁇ 4, preferably ⁇ 3, 2, 1, more preferably 0;
  • the sequence of the core fragment is selected from one or more of SEQ ID NO.: 1-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15; and
  • the short peptide has an activity of binding to an anti-HPV16 type E6-E7 protein antibody
  • the peptide set contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 selected from the group consisting of Formula I a short peptide or a polypeptide derived therefrom, preferably, the peptide set contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 selected from the group consisting of SEQ ID NO.
  • the peptide set further comprises an isolated HPV epitope peptide other than SEQ ID NO.: 1-15, preferably an HPV 16 type E6-7 protein epitope peptide.
  • the peptide set contains at least the short peptide represented by SEQ ID NO.: 5 and/or SEQ ID NO.: 14.
  • X is a core fragment of 4-8 amino acids; preferably 4, 5, 6, 7, or 8 amino acids;
  • X1, X2 are none, 1, 2 or 3 amino acids, and the total number of X1 and X2 amino acids is ⁇ 4, preferably ⁇ 3, 2, 1, more preferably 0;
  • the sequence of the core fragment is selected from one or more of SEQ ID NO.: 1-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15; and
  • the short peptide has an activity of binding to an anti-HPV16 type E6-E7 protein antibody
  • the isolated short peptide is derived from human papillomavirus (HPV) type 16 E6-7 protein.
  • said X1 or X2 corresponds to a flanking amino acid sequence of said core fragment X in a human papillomavirus (HPV) type 16 E6-7 protein.
  • the short peptide has a length of ⁇ 10 amino acids, preferably ⁇ 9 amino acids, more preferably ⁇ 8 amino acids.
  • the short peptide is selected from the group consisting of short peptides as shown in SEQ ID NO.: 1-15.
  • the short peptide satisfying Formula I is also shown in Table 2.
  • an isolated derivative polypeptide having the core fragment of the second aspect of the invention wherein the polypeptide is X1 or X2 of the short peptide of the second aspect of the invention, respectively A polypeptide having 1-3 amino acid additions, 1-2 deletions and still containing the core fragment X, and having binding activity to an anti-human HPV16 type E6-7C antibody.
  • said X1 or X2 is an arbitrary sequence, respectively, and is not a flanking amino acid sequence of said core fragment X in a human papillomavirus (HPV) type 16 E6-E7 protein.
  • HPV human papillomavirus
  • an expression vector expressing the short peptide of the second aspect of the invention or a derivative thereof, or the peptide set of the first aspect of the invention.
  • fusion carrier protein having the following structure:
  • Carrier protein - a polypeptide of formula I.
  • the carrier protein comprises GST.
  • the carrier protein comprises truncated GST188 (consisting of 188 amino acid residues).
  • the use of the short peptide or the derivative thereof of the second aspect of the invention, the peptide assembly of the first aspect of the invention, or the fusion carrier protein of the fifth aspect of the invention characterized in that It is used to prepare reagents and kits for diagnosing or detecting human papillomavirus (HPV).
  • HPV human papillomavirus
  • the human papillomavirus is of the HPV type 16.
  • kits comprising a peptide or a derivative thereof according to the second aspect of the present invention, a peptide set according to the first aspect of the present invention, or a fifth of the present invention.
  • the instructions describe that the detection reagent in the kit is used to diagnose or detect human papillomavirus (HPV).
  • the kit further comprises the short peptide of the second aspect of the invention or a derivative thereof, or the peptide set of the first aspect of the invention as a positive control.
  • the eighth aspect of the present invention provides the use of the short peptide or the derivative polypeptide thereof according to the second aspect of the present invention, the peptide assembly of the first aspect of the present invention, or the fusion protein of the fifth aspect of the present invention, (a) For the preparation of a pharmaceutical composition for preventing and/or treating human papillomavirus; (b) as an antigen for preparing a detection reagent; and/or (c) for use as an immunogen for the preparation of a therapeutic drug.
  • the pharmaceutical composition comprises the short peptide of the second aspect of the invention or a derivative thereof, the peptide set of the first aspect of the invention, or the fusion protein of the fifth aspect of the invention And a pharmaceutically acceptable carrier.
  • the medicament comprises a vaccine composition.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the short peptide of the second aspect of the invention or a derivative thereof, the peptide set of the first aspect of the invention, or the fifth aspect of the invention
  • the fusion protein described and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine composition.
  • the vaccine composition is monovalent or multivalent.
  • a method for non-diagnostic determination of whether a sample contains human papillomavirus HPV in vitro characterized in that the short peptide or the derivative polypeptide thereof according to the second aspect of the present invention in the test sample, the present invention
  • the peptide set, or the fusion protein of the fifth aspect of the invention has an expression level E1, and the short peptide of the second aspect of the invention or a derivative thereof thereof in the normal sample, the peptide of the first aspect of the invention
  • the expression or the expression level E0 of the fusion protein according to the fifth aspect of the present invention is compared. If E1 ⁇ 2E0, the sample contains human papillomavirus HPV.
  • the HPV is of the HPV type 16.
  • a method for diagnosing human papillomavirus wherein the short peptide of the second aspect of the present invention or a polypeptide derived therefrom, the peptide set of the first aspect of the present invention, or the like, Or the expression level E1 of the fusion protein according to the fifth aspect of the invention is detected, and compared with the expression level E0 of the short peptide of the second aspect of the invention or the peptide set of the first aspect of the invention in a normal sample, if E1 ⁇ 2E0 indicates that the test subject is infected with human papillomavirus HPV.
  • a method for preventing and/or treating human papillomavirus which comprises administering the short peptide of the second aspect of the present invention, the peptide set of the first aspect of the present invention or the present invention to a subject in need thereof.
  • Figure 1 Immunoblot identification of the first round of antigenic peptide mapping of HPV16 E6 protein.
  • Figure 1A shows a set of 16-mer peptide (E6/P1 - P19) immunoblot identifications spanning 9 residues across the full-length HPV16 E6 protein sequence;
  • Figure 1B shows immunoblot identification using pre-immune rabbit control sera.
  • Primary antibody is a 1:2000 dilution of rabbit anti-recombinant E6-E7 protein antiserum. Although there were 10 16-reactive peptides in P1-P2, P5-P6, P8, P12, P14, P16-P17 and P19 in the Western blotting experiment (Fig. 1A), the P16 peptide was also recognized in the pre-immune rabbit control sera. Fig. 1B), so it is excluded that it is an antigenic peptide specific for HPV16 E6.
  • Figure 2 shows a set of 16-mer peptide (E7/P1–P12) immunoblot identifications spanning 9 residues across the full-length HPV16 E7 protein sequence.
  • the primary antibody used rabbit anti-recombinant E6-E7 protein antiserum diluted 1:2000.
  • 16 polypeptides P1-P6 and P12 are imprinted reactive antigenic peptides. Since no reactive bands were produced with the pre-immune rabbit control serum, the results were not shown.
  • Figure 3 shows the immunoblot identification of the HPV16 E6-2 fine epitope peptide.
  • Figure 3A shows a set of 8-polypeptide (E6/P37-P43) immunoblots covering a full-length P5-reactive 16-mer peptide with 7 residues overlapping each other;
  • Figure 3B based on the consensus sequence of residues in the reactive 8 peptide Epitope minimum motif analysis. The shaded portion indicates the peptide sequence common to the reactive peptide.
  • Figure 4 shows the immunoblot identification of the HPV16 E6-3 fine epitope peptide.
  • Figure 4A shows a set of 8-mer peptide (E6/P44-P50) immunoblots covering a full-length P6-reactive 16-mer peptide with 7 residues overlapping each other;
  • Figure 4B based on the consensus sequence of residues in the reactive 8 peptide Epitope minimum motif analysis. The shaded portion indicates the peptide sequence common to the reactive peptide.
  • FIG. 5 shows the immunoblot identification of the HPV16 E7-3 epitope peptide.
  • 5A a set of 8-polypeptide (E7/P29-P37) covering a 7-residue overlap of P3 reactive peptides was identified by immunoblotting;
  • FIG. 5B based on the minimal base of the consensus residue sequence in the reactive 8 peptide Sequence analysis. The shaded portion indicates the peptide sequence common to the reactive peptide.
  • Figure 6 shows the immunoblot identification of the HPV16 E7-4 epitope peptide.
  • Figure 6A covering the full length P4 A group of 8-polypeptides (E7/P38-P44) with 16 residues overlapping each other were identified by immunoblotting;
  • Figure 6B based on the minimal motif analysis of the consensus residues in the reactive 8 peptide. The shaded portion indicates the peptide sequence common to the reactive peptide.
  • the present inventors have firstly identified 15 small molecule epitope peptides on human papillomavirus (HPV) by extensive and intensive research using a modified biosynthetic peptide method, and this group of epitope peptides is passed as a minimal motif peptide. Detection of antibodies induced by multiple epitopes on the target protein increases the sensitivity of clinical detection, increases the dilution of the test serum sample, thereby reducing the interference of many low levels of anti-unknown antigen antibodies in the body, greatly reducing or completely avoiding False positive test results. On the basis of this, the present invention has been completed.
  • epitope polypeptide of the present invention refers to a short peptide conforming to the structural formula of formula I or containing a core sequence X.
  • a generic term for an epitope polypeptide refers to a short peptide conforming to the structural formula of formula I or containing a core sequence X.
  • epitopope short peptide conforming to the formula I formula and “short peptide of the present invention” are used interchangeably and refer to a short peptide which conforms to the following formula and which shows a short peptide which satisfies the following characteristics:
  • X is a core fragment of 4-8 amino acids; preferably 4, 5, 6, 7, or 8 amino acids;
  • X1 and X2 are none, 1 or 2 amino acids, and the sum of the number of X1 and X2 amino acids is ⁇ 2;
  • the sequence of the core fragment is selected from one or more of SEQ ID NO.: 1-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15; and
  • the short peptide has an activity of binding to an anti-HPV16 type E6-7 protein antibody
  • motif peptide refers to a core fine fragment of the smallest 6-7 amino acids that can be recognized by an anti-HPV antibody on the HPV type 16 E6-E7 protein. , that is, X in Formula I.
  • polypeptide containing core sequence X and “polypeptide of the invention” are used interchangeably. It should be understood that The term also includes short peptides conforming to Formula I and derivatives of polypeptides containing core sequence X, meaning that short peptides conforming to the structural formula of Formula I are added after 1-3 amino acids, 1-2 deletions and still contain the core fragment X
  • the sequence has a polypeptide that binds to an anti-human HPV 16 antibody.
  • conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • peptide set refers to a peptide set consisting of a peptide or polypeptide selected from the group consisting of at least two selected from the short peptides of the invention or derived polypeptides thereof.
  • the peptide set of the present invention contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 selected from the second aspect of the invention. a short peptide or a polypeptide derived therefrom; more preferably, the peptide set contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 selected from the group consisting of SEQ ID NO.: a short peptide of 1-15 or a polypeptide derived therefrom. Furthermore, the peptide set further comprises an isolated HPV epitope peptide other than SEQ ID NO.: 1-15, preferably an HPV 16 type E6-7 protein epitope peptide.
  • the peptide set comprises at least the short peptide represented by SEQ ID NO.: 5 and/or SEQ ID NO.: 14, preferably, the peptide set may also be divided into the above two
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 are selected from the sequence consisting of SEQ ID NO.: 1-15.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • a polypeptide in a natural state in a living cell is not isolated and purified, but the same polypeptide is isolated and purified if it is separated from other substances existing in the natural state.
  • isolated peptide means that the polypeptide of the invention is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify the polypeptides of the invention using standard protein purification techniques.
  • a substantially purified polypeptide (fusion protein) produces a single major band on a non-reducing polyacrylamide gel.
  • the polypeptide of the present invention comprises a short peptide conforming to the structural formula of formula I or a polypeptide comprising core sequence X.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • recombinant peptides can be used to obtain the relevant peptide sequences in large quantities. This is usually carried out by cloning into a vector, transferring it to a cell, and then separating the related peptide (fusion protein) from the proliferated host cell by a conventional method.
  • HPV Human papillomavirus
  • HPV Human papillomavirus
  • HPV16 Human papillomavirus
  • HPV HPV is weaker than other viruses (lower antibody response level), especially unlike other viruses, there is a self-clearing mechanism within 6 months to two years, and it is still unclear when antibodies occur.
  • the commonly used E6, E7 and L1 diagnostic antigens or their antigenic peptides lack real specificity, and the accuracy of serological detection is not high.
  • HPV targeted by the present invention is mainly HPV16 type virus, and the genome sequence GenBank No.: K02718.1.
  • sequences of oncogenic proteins E6 and E7 are shown in SEQ ID NO.: 16 and SEQ ID NO.: 17, respectively.
  • the HPV16 type virus used in the present invention can be obtained and sequenced, amplified or expressed by a conventional method.
  • a preferred method is as follows: according to the HPV16 prototype viral genome sequence (GenBank No.: K02718.1), a serial expression gene encoding a full-length E6 (158aa) and E7 (98aa) protein is chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd. The gene sequence was confirmed by DNA sequencing, and it was recombined into the prokaryotic expression plasmid of pRSET-A by conventional molecular cloning techniques.
  • pXXGST-1 fusion expression vector Xu et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide
  • Biosynthesis strategy J Reprod Immunol, 81:9-16, 2009. Therefore, first use DNA recombination technology (via chemistry) Synthesizing the positive and negative strands of the coding DNA, annealing and inserting the expression plasmid, transforming the E. coli BL21 (DE3) host strain, verifying the inserted DNA sequence by recombinant cloning, and thermally inducing the expression of the GST188-16 polypeptide fusion protein.
  • a series of fusion expression expression 8 peptides (E6/P20 ⁇ P94, E7/P13 ⁇ P68) expressing GST188 as a vector on the overlapping 6 residues of HPV16 type E6 and E7 proteins on the HPV16 type E6 and E7 proteins were further constructed.
  • the rabbit anti-E6-E7 polyclonal antibody was used to identify the epitopes that may be present in all reactive peptides displayed by the first round of antigenic peptide scanning and the antibody recognition minimal motif.
  • eight fine epitope peptides were identified on the E6 protein based on the amino acid sequence common between a plurality of 8 peptides positive for each epitope peptide imprinting reaction.
  • E6-1 The epitope is located in the P1 and P2 overlap regions; seven epitope peptides are identified on the E7 protein, which are TLHEYML 7-13 , MLDLQ 12-17 , DLYC 21-24 , DSSEE 30-34 , DEIDGP 36-41 , AGQAEP 42-47 (should be 42-47) and SQKP 95-98 (E7-2 and E7-6 are located in the overlap of P1 and P2 and P5 and P6, respectively).
  • the identification of 15 minimal motif BCE peptides of HPV16 of the present invention can be used for serological diagnosis of HPV, and a therapeutic HPV multi-epitope peptide vaccine (mainly T cell epitope for inducing cellular immunity) is designed and designed for future. And laid the foundation for the preparation of antibodies for each epitope used in basic research.
  • a therapeutic HPV multi-epitope peptide vaccine mainly T cell epitope for inducing cellular immunity
  • HPV minimal motif epitope peptides of the present invention are chemically synthesized either alone or in combination, or are fused to the GST protein to form a fusion carrier protein, which can be used as an ELISA, peptide for developing antibodies for detecting HPV16 virus-infected antibodies.
  • a fusion carrier protein which can be used as an ELISA, peptide for developing antibodies for detecting HPV16 virus-infected antibodies.
  • the use of peptide antigens by microarray and Western blotting has helped to establish a serological assay for HPV16 infection that can be applied to clinical diagnostics and epidemiological studies.
  • the minimal motif peptide of the present invention can be used as an immunogen, and after administration to a subject in need thereof, T cell reactivity can be stimulated, thereby preventing and/or treating HPV infection.
  • the present invention provides a method for diagnosing HPV (especially type 16) using an epitope peptide. These tests are well known in the art. Anti-HPV protein antibodies are tested in the test and can be used to diagnose HPV infection.
  • the method for detecting the presence or absence of an anti-HPV protein antibody in a sample can be detected by the principle that the polypeptide of the present invention specifically binds to HPV in the sample, and comprises: reacting the sample with the antigen of the polypeptide of the present invention or a fusion-expressing protein thereof, and detecting the detection Whether the sample OD value is twice as large as the control, or whether the antibody complex (imprint strip) is formed, the OD value is high or the imprinted band is formed to indicate the presence of an antibody against the HPV protein antibody or an epitope in the sample.
  • the epitope peptide (or peptide collection) of the present invention can be fixedly spotted on a protein chip to form a reagent or kit for detecting an anti-HPV protein antibody in the sample.
  • polypeptide of the invention (chemically synthesized peptide, or a fusion protein conjugated to a carrier protein, or by construction of a recombinant multi-epitope peptide antigen) can be spotted on a test plate or test strip.
  • the sample to be tested is immunologically reacted with the polypeptide of the present invention, thereby diagnosing whether or not the human HPV antibody and/or the antibody of the human HPV epitope are present in the sample to be tested.
  • the test plate can be made by using a conventional test plate preparation method using a test plate material commonly used in the art.
  • the representative immunoassay plate comprises a test strip and a support plate supporting the test strip, such as a PVC polyester plate, etc.; the test strip is composed of a filter paper, a chromatography material, a nitrocellulose membrane and an absorbent paper.
  • the lap joint may be fixedly connected by a conventional method such as tape; wherein: the chromatographic material is pre-coated with colloidal gold or a colored label of the polypeptide or peptide set of the present invention, preferably with the peptide of the present invention conjugated with a carrier protein
  • the collection is performed, and the chromatographic material is pre-coated with colloidal gold, and the detection line and the quality control line are adsorbed on the nitrocellulose membrane.
  • the invention also provides a pharmaceutical composition.
  • the pharmaceutical compositions of the invention may be therapeutic or prophylactic (e.g., vaccines).
  • the pharmaceutical composition of the present invention comprises an effective amount of an epitope peptide or peptide set of the present invention, or dendritic cells sensitized with the short peptide or T cells induced with dendritic cells, and at least one pharmaceutically acceptable Accepted carrier, diluent or excipient.
  • these (vaccine) compositions comprise an immunological antigen (including the short peptides of the present invention, a peptide set or a derivative thereof), and are usually combined with a "pharmaceutically acceptable carrier" including those which are not induced by themselves. Any carrier that is harmful to an individual receiving the composition.
  • suitable carriers include, but are not limited to, proteins, lipid agglutins (such as oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. Additionally, these carriers can function as immunostimulating agents ("adjuvants").
  • compositions of the invention may also contain additional adjuvants.
  • vaccine adjuvants include (but Not limited to the following types: inorganic adjuvants such as aluminum hydroxide, alum, etc.; synthetic adjuvants such as synthetic double-stranded polynucleotides (double-stranded polyadenylation, uridine), levamisole , isoproterenol, etc.; oil agents, such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil and so on.
  • the vaccine composition or immunogenic composition can be formulated as an injectable, such as a liquid solution or suspension; it can also be formulated in a solid form suitable for solution or suspension, liquid excipient prior to injection.
  • the formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
  • composition can be formulated in unit or multi-dose form.
  • Each dosage form contains a predetermined amount of active material calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.
  • compositions can be administered by conventional routes including, but not limited to, intravenous, intratumoral, intramuscular, intraperitoneal, subcutaneous, intradermal, paracancerous, or topical administration.
  • a safe and effective amount of an epitope peptide or peptide set of the invention is administered to a human, wherein the safe and effective amount is typically at least about 1 microgram of peptide per kilogram of body weight, and in most cases no more than about 8 mg of peptide per kilogram of body weight, preferably the dose is from about 1 microgram to 1 milligram of peptide per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention identifies a set of minimal motif epitope peptides on HPV type 16 E6 and E7 proteins that specifically bind to anti-human HPV antibodies.
  • the invention constructs a truly specific detection antigen, and improves the sensitivity of clinical detection by detecting antibodies induced by multiple epitopes on the target protein, which can increase the dilution of the test serum sample, thereby reducing the number of low in vivo Levels of interference against unidentified antigenic antibodies (reducing their detection sensitivity as dilution increases) greatly reduce or completely avoid false positive test results.
  • a vaccine composition comprising the minimal motif peptide of the present invention is expected to be used for active immunization of high-risk subjects, by stimulating T cells to generate an immune response, and to achieve the effect of preventing and/or treating HPV.
  • Example 1 Scanning of the first round of antigenic peptides of HPV16 E6 and E7 proteins
  • HPV16 E6 and E7 protein tandem coding genes were chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd. according to the prototype HPV16 genome sequence (GenBank No.: K02718.1).
  • the prokaryotic expression plasmid pBV21 was kindly provided by Professor Sun Fangwei from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the pRSET C plasmid was purchased from Invitrogen, USA.
  • the heat-induced expression plasmids pXXGST-1 and pXXGST-2 were constructed by the inventors of the present invention (Patent No.: ZL 200710173305.2).
  • Escherichia coli BL21 (DE3) strain was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.
  • Restriction enzymes EcoR I, BamH I, Sal I, Taq enzyme and T4 DNA ligase were purchased from TaKaRa Biotechnology, Japan, pre-stained protein molecular weight standard, horseradish peroxidase-labeled goat anti-rabbit secondary antibody (IgG/ HRP), diaminobenzidine (DAB) and 0.2 ⁇ m nitrocellulose membrane were purchased from a German company (Whatman GmbH, Dossel, Germany) represented by Shanghai Biotech Biotechnology Services. 6 ⁇ His monoclonal antibody was purchased from Aogma, USA (Cat. No.: 9618).
  • QIAprep spin miniprep Kit Plasmid extraction kit, QIAquick PCR product purification kit and quick gel recovery kit were purchased from QIAGEN, Germany.
  • New Zealand white rabbits were purchased from Shanghai BK Laboratory Animals Co., Ltd. EcoR I and Sal I and BamH I and EcoR I
  • the preparation process is summarized as follows: 1) expression of recombinant HPV16 E6-E7 fusion protein by IPTG induction; 2) purification of two target proteins by preparative gel electrophoresis; 3) purification of recombinant E6-E7 fusion protein as immunogen, After the emulsification of Freund's adjuvant, 4 male New Zealand white rabbits were actively immunized; 4) After the booster immunization, the serum was taken and stored in a refrigerator at -20 °C for use.
  • the recombinant E6 and E7 short peptide clones with the correct sequencing result of the insert were inoculated into 3 ml of LB culture medium of Amp, shake cultured at 30 ° C overnight, and transferred to fresh Amp-containing LB medium at a ratio of 1/50. After shaking at 30 °C for 2 to 3 hours until 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 for use; the expression of the E6-E7 clone was added. The cells were cultured overnight at 37 ° C, and transferred to fresh LB-containing LB medium at a ratio of 1/50 on the morning of the morning, shake culture at 37 ° C, and induced by IPTG for 4 h.
  • the induced total bacterial protein samples were simultaneously subjected to 15% SDS-PAGE. After electrophoresis, one gel was stained with Coomassie brilliant blue, and the two gels were transferred to nitrocellulose membrane (100 mA) for 2 h, using Li Chunhong. The membrane was stained for 2 min, and the needle was punched with a needle at the band of the 16-mer short peptide fusion protein, and the Ponceau red stain was washed off with water;
  • the blotting membrane was washed repeatedly four times with PBS buffer, blocked with 5% skim milk powder overnight, and washed four times with PBS buffer.
  • Add 30 ⁇ l of anti-rabbit anti-recombinant E6-E7 fusion protein serum to 8-10 ml of reaction solution or Rabbit serum was reacted at room temperature for 2 h, washed with PBS buffer, and then added with 5 ⁇ l of secondary anti-rabbit anti-rabbit IgG/HRP (1:10000 dilution), reacted at room temperature for 1 h, washed with PBS buffer and developed with ECL chemiluminescence kit (Fig. 1 and Figure 2).
  • Example 2 Identification of the minimal epitope of two representative antigenic peptides in the second round of HPV16 E6 protein
  • P5 and P6 reactive peptides were used as examples to identify their minimal recognition of antibody recognition.
  • a series 8 peptide spanning 7 amino acid residues spanning the full length sequence of P5 and P6 is designed to encode a positive and negative strand of DNA (positive strand 5'-end plus 5'-gatcc, 3'- The taag-3' was added at the end; the 5'-end of the minus strand was added with 5'-tcgactta, and the 3'-end was added with g-3'), and the DNA fragment was synthesized.
  • the 2-4 operation steps are the same as the 2-4 steps in Example 1.
  • the blotting membrane was washed repeatedly four times with PBS buffer, blocked with 5% skim milk powder overnight, and washed four times with PBS buffer, and 30 ⁇ l of anti-rabbit anti-human recombinant E6-E7 protein serum was added to 8-10 ml of the reaction solution. 1:2000 dilution), react at room temperature for 2 h, wash with PBS buffer, add 5 ⁇ l of secondary anti-human anti-human IgG/HRP (1:10000 dilution), react at room temperature for 1 h, wash with PBS buffer and perform ECL chemiluminescence (Fig. 3A and Figure 4A).
  • Example 3 Identification of the minimal epitope of two representative antigenic peptides in the second round of HPV16 E7 protein
  • the 2-4 operation steps are the same as the 2-4 steps in Example 1.
  • the blotting membrane was washed repeatedly four times with PBS buffer, blocked with 5% skim milk powder overnight, and washed four times with PBS buffer, and 30 ⁇ l of anti-rabbit anti-human recombinant E6-E7 protein serum was added to 8-10 ml of the reaction solution. 1:2000 dilution), react at room temperature for 2 h, wash with PBS buffer, add 5 ⁇ l of secondary anti-human anti-human IgG/HRP (1:2000 dilution), react at room temperature for 1 h, wash with PBS buffer and perform ECL chemiluminescence (Fig. 5A and Figure 6A).

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Abstract

本发明公开了十五个人乳头瘤病毒(HPV)16型E6和E7蛋白的抗原表位最小基序肽，其可用于制备识别HPV16 E6和E7蛋白单或多克隆抗体，特别是可单独或组合制备专门用于检测HPV16病毒感染后所生成抗体的化学合成肽和/或重组多表位肽抗原。

Description

人乳头瘤病毒的表位肽及其应用 技术领域
本发明属于生物工程和免疫学技术领域，具体地，涉及与妇女宫颈癌发生最密切相关的人乳头瘤病毒(HPV)16型E6和E7致癌蛋白能诱发抗体的全部精细线性B细胞表位肽及其应用。
背景技术
众所周知，一靶蛋白上能诱发抗体生成的B细胞表位(B cell epitope,BCE)肽，能用作研制抗体有效的预防性/治疗性合成肽疫苗的候选表位肽，或用来制备科学研究和临床诊断用的单克隆或多克隆抗体(单抗或多抗)。因此，通常使用成熟的常规化学合成肽法、噬菌体展示文库法、不同长短肽芯片法以及借助计算机软件预测鉴定已知氨基酸序列靶蛋白上的BCE肽。
但是，由于表位扫描作图方法的限制，以往获得可用于合成肽疫苗和临床诊断肽抗原或抗体Kit研制的肽段经常都是大于8个残基的肽段，即它们实际上都是非抗体识别的最小序列肽，都是含一特异表位基序的一段抗原性肽，因而不能真正称为BCE肽。这样的抗原性肽在实际应用上的缺陷是显而易见的，也就是，肽越长，其中的N个表位数越多。因而作为合成肽疫苗的候选表位肽，其游离母蛋白抗原后单独诱发特定功能性/保护性抗体的效果，显然存在不确定性或效果不能达到最佳(因为在研究疫苗的这一抗原性肽内的N个表位中，哪一个潜在的表位是诱发抗体的优势表位就无法知晓或确定)。其作为诊断肽抗原往往也存在缺乏特异性的问题，尤其在自身免疫疾病和病毒自然感染诱发抗体滴度不高的情况下，由于检测血清样本稀释度通常不可能高(常见1:10-1:100)，加上人体内原本就存在成千上万种抗不明抗原的抗体这一因素，因而易出现一些或较高的假阳性检测结果就能理解了。特别需要指出的是，许多病毒感染或自身免疫疾病抗体检测不容许假阳性结果，因为那会因此对检测的正常对象带来不必要的药物治疗伤害和无谓甚至高昂的经济负担。另外，这样的检测也会导致许多临床基础研究结果的不确定性。
人类乳头状瘤病毒(human papillomavirus,HPV)是一组环状DNA病毒的总称。目前发现HPV型别已超过150多种，其中超过40种型别可感染女性生殖道上皮，而与宫颈癌以及宫颈上皮内高度病变发生密切相关的高危型HPV涉及HPV16、 18、26、31、33、35、39、45、52、53、56、58、66、67、69、73、82和97等20多种型别，其中，HPV16流行率在世界各国排第一。
现已清楚高危型HPV导致的子宫颈癌在妇女肿瘤发生中位居第三位。作为针对严重威胁妇女生殖健康的HPV病毒的预防和治疗措施，准确诊断不同高危型HPV感染是关键的第一步。目前的诊断方法可主要分为三大类，即细胞学诊断、HPV DNA鉴定和HPV血清学检测。其中前两者已在临床中普遍应用，后者经济简便但目前仍然始终处于研究中。究其原因，就是相对其他病毒HPV免疫原性弱(感染后抗体应答水平低)，而目前普遍采用的E6、E7和L1诊断抗原或它们的抗原性肽又缺乏真正的特异性，乃至检测准确率不高。
鉴于以上所说技术发展背景，有必要建立新型高特异性高灵敏度的HPV诊断用重组多表位肽检测抗原。
发明内容
本发明提供了一种人乳头瘤病毒的表位肽及其应用。
本发明第一方面，提供了一种分离的肽集合，所述的肽集合由选自下组的肽或多肽构成：至少两种选自式I所述的短肽或其衍生多肽，其中
X1-X-X2
式I，
并满足以下特征：
(a)式I中X为4-8个氨基酸的核心片段；较佳地为4、5、6、7、或8个氨基酸；
(b)式I中X1、X2为无、1、2或3个氨基酸，且X1和X2氨基酸个数总和≤4，较佳地，≤3、2、1，更佳地为0；
(c)所述的核心片段的序列选自SEQ ID NO.:1-15中的一个或多个，较佳地为1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15个；和
(d)所述的短肽具有与抗HPV16型E6-E7蛋白抗体结合的活性；
其中，“-”表示肽键或肽接头。
在另一优选例中，所述的肽集合中至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自式I所述的短肽或其衍生多肽，优选地，所述肽集合至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自SEQ ID NO.:1-15的短肽或其衍生多肽。
在另一优选例中，所述的肽集合还包括除SEQ ID NO.:1-15以外的分离的HPV表位肽，优选地，为HPV 16型E6-7蛋白表位肽。
在另一优选例中，其特征在于，所述的肽集合中至少含有SEQ ID NO.:5和/或SEQ ID NO.:14所示的短肽。
本发明第二方面，提供了一种分离的短肽，所述短肽的结构如式I所示：
X1-X-X2
式I，
并满足以下特征：
(a)式I中X为4-8个氨基酸的核心片段；较佳地为4、5、6、7、或8个氨基酸；
(b)式I中X1、X2为无、1、2或3个氨基酸，且X1和X2氨基酸个数总和≤4，较佳地，≤3、2、1，更佳地为0；
(c)所述的核心片段的序列选自SEQ ID NO.:1-15中的一个或多个，较佳地为1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15个；和
(d)所述的短肽具有与抗HPV16型E6-E7蛋白抗体结合的活性；
其中，“-”表示肽键或肽接头。
在另一优选例中，所述分离的短肽来自人乳头瘤病毒(HPV)16型E6-7蛋白的。
在另一优选例中，所述X1或X2分别对应于所述核心片段X在人乳头瘤病毒(HPV)16型E6-7蛋白中侧翼氨基酸序列。
在另一优选例中，所述的短肽的长度≤10个氨基酸，较佳地≤9个氨基酸，更佳地≤8个氨基酸。
在另一优选例中，所述的短肽选自如SEQ ID NO.:1-15所示的短肽。
在另一优选例中，所述满足式I的短肽还如表2所示。
表2
Figure PCTCN2015090776-appb-000001
Figure PCTCN2015090776-appb-000002
本发明第三方面，提供了一种具有本发明第二方面所述核心片段的分离的衍生多肽，其特征在于，所述多肽是经本发明第二方面所述的短肽的X1或X2分别经过1-3个氨基酸的添加、1-2个缺失并仍含有所述核心片段X，且具有与抗人HPV16型E6-7C抗体结合活性的多肽。
在另一优选例中，所述X1或X2分别是任意的序列、且不是所述核心片段X在人乳头瘤病毒(HPV)16型E6-E7蛋白中侧翼氨基酸序列。
本发明第四方面，提供了一种表达载体，所述的表达载体表达本发明第二方面所述的短肽或其衍生多肽、或本发明第一方面所述的肽集合。
本发明第五方面，还提供了一种融合载体蛋白，所述的融合载体蛋白具有以下结构：
式I短肽-载体蛋白；或
载体蛋白-式I多肽。
在另一优选例中，所述的载体蛋白包括GST。
在另一优选例中，所述的载体蛋白包括截短GST188(由188个氨基酸残基组成)。
本发明第六方面，提供了本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述的肽集合、或本发明第五方面所述的融合载体蛋白的用途，其特征在于，用于制备诊断或检测人乳头瘤病毒(HPV)的试剂和试剂盒。
在另一优选例中，所述的人乳头瘤病毒为HPV16型。
本发明第七方面，提供了一种试剂盒，所述的试剂盒含有特异性检测本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合或本发明第五方面所述的融合蛋白的检测试剂，和说明书。
在另一优选例中，所述的说明书中记载：该试剂盒中的检测试剂用于诊断或检测人乳头瘤病毒(HPV)。
在另一优选例中，所述的试剂盒还含有本发明第二方面所述短肽或其衍生多肽、或本发明第一方面所述肽集合作为阳性对照。
本发明第八方面，提供了本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白的用途，(a)用于制备预防和/或治疗人乳头瘤病毒的药物组合物；(b)作为抗原用于制备检测试剂；和/或(c)作为免疫原用于制备治疗药物。
在另一优选例中，所述的药物组合物中含有本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白，和药学上可接受的载体。
在另一优选例中，所述药物包括疫苗组合物。
本发明第九方面，提供了一种药物组合物，所述组合物含有本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白，和药学上可接受的载体。
在另一优选例中，所述的药物组合物为疫苗组合物。
在另一优选例中，所述疫苗组合物为单价或多价。
本发明第十方面，提供了一种体外非诊断性确定样本是否含有人乳头瘤病毒HPV的方法，其特征在于，检测样本中本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白的表达量E1，并与正常样本中本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白的表达量E0进行比较，若E1≥2E0，则说明该样本中含有人乳头瘤病毒HPV。
在另一优选例中，所述HPV为HPV 16型。
本发明第十一方面，提供了一种诊断人乳头瘤病毒的方法，对来自检测对象的样本中本发明第二方面所述短肽或其衍生多肽、本发明第一方面所述肽集合、或本发明第五方面所述的融合蛋白的表达量E1进行检测，并与正常样本中本发明第二方面所述短肽或本发明第一方面所述肽集合的表达量E0进行比较，若E1≥2E0，则说明该检测对象感染人乳头瘤病毒HPV。
本发明第十二方面，提供了一种预防和/或治疗人乳头瘤病毒的方法，向需要的对象施用本发明第二方面所述短肽、本发明第一方面所述肽集合或本发明第九方面所述 的药物组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.HPV16 E6蛋白第一轮抗原性肽作图的免疫印迹鉴定。图1A显示了一组跨越全长HPV16 E6蛋白序列相互重叠9个残基的16聚肽(E6/P1–P19)免疫印迹鉴定；图1B显示了使用免疫前兔对照血清的免疫印迹鉴定。
注：一抗是1:2000稀释的兔抗重组E6-E7蛋白抗血清。虽然蛋白印迹试验中出现P1-P2、P5-P6、P8、P12、P14、P16-P17和P19共10个16聚反应性肽(图1A)，但因为免疫前兔对照血清也识别P16肽(图1B),所以排除其为HPV16 E6特异的抗原性肽。
图2显示了一组跨越全长HPV16 E7蛋白序列相互重叠9个残基的16聚肽(E7/P1–P12)免疫印迹鉴定。一抗使用1:2000稀释的兔抗重组E6-E7蛋白抗血清。图中16聚肽P1-P6和P12为印迹反应性抗原性肽。因为用免疫前兔对照血清未产生任一反应性条带，所以结果未显示。
图3显示了HPV16 E6-2精细表位肽的免疫印迹鉴定。其中图3A，覆盖全长P5反应性16聚肽相互重叠7个残基的一组8聚肽(E6/P37-P43)免疫印迹鉴定；图3B，依据反应性8肽中共有残基序列的表位最小基序分析。阴影部分表示反应性肽中共同的肽序列。
图4显示了HPV16 E6-3精细表位肽的免疫印迹鉴定。其中图4A，覆盖全长P6反应性16聚肽相互重叠7个残基的一组8聚肽(E6/P44-P50)免疫印迹鉴定；图4B，依据反应性8肽中共有残基序列的表位最小基序分析。阴影部分表示反应性肽中共同的肽序列。
图5显示了HPV16 E7-3表位肽的免疫印迹鉴定。其中图5A，覆盖P3反应性肽相互重叠7个残基的一组8聚肽(E7/P29-P37)免疫印迹鉴定；图5B，依据反应性8肽中共有残基序列的表位最小基序分析。阴影部分表示反应性肽中共同的肽序列。
图6显示了HPV16 E7-4表位肽的免疫印迹鉴定。其中图6A，覆盖全长P4反 应性16聚肽相互重叠7个残基的一组8聚肽(E7/P38-P44)免疫印迹鉴定；图6B，依据反应性8肽中共有残基序列的表位最小基序分析。阴影部分表示反应性肽中共同的肽序列。
具体实施方式
本发明人通过广泛而深入的研究，运用改良生物合成肽的方法，首次鉴定了人乳头瘤病毒(HPV)上的15个小分子表位肽，这组表位肽作为最小基序肽，通过检测暴露在靶蛋白上多个表位诱发的抗体而提高临床检测的灵敏度，可增加检测血清样本的稀释度，由此可减少体内众多低水平抗不明抗原抗体的干扰，极大地降低或完全避免假阳性检测结果。在此基础上，完成了本发明。
在本发明中，“本发明表位肽”、“本发明多肽”、“本发明活性片段”、“最小基序肽”可互换使用，指符合式I结构式的短肽或含有核心序列X的表位多肽的统称。
术语“符合式I结构式的表位短肽”、“本发明短肽”可互换使用，指符合下式且所示的短肽满足以下特征的短肽：
X1-X-X2
式I，
并满足以下特征：
(a)式I中X为4-8个氨基酸的核心片段；较佳地为4、5、6、7、或8个氨基酸；
(b)式I中X1、X2为无、1或2个氨基酸，且X1和X2氨基酸个数总和≤2；
(c)所述的核心片段的序列选自SEQ ID NO.:1-15中的一个或多个，较佳地为1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15个；和
(d)所述的短肽具有与抗HPV16型E6-7蛋白抗体结合的活性；
其中，“-”表示肽键或肽接头。
在本发明中，一种优选的符合式I结构式的短肽序列如SEQ ID NO.:1-15所示。
术语“基序肽”、“表位肽”、“核心片段”可互换使用，均指HPV 16型E6-E7蛋白上可为抗HPV抗体识别结合的最小6-7个氨基酸的核心精细片段，即式I中的X。
术语“含有核心序列X的多肽”、“本发明多肽”可互换使用。应理解，所述 术语还包括符合式I的短肽以及含有核心序列X的多肽的衍生物，指符合式I结构式的短肽在经过1-3个氨基酸添加、1-2个缺失并仍含有所述核心片段X的序列，且具有与抗人HPV 16抗体结合活性的多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
Figure PCTCN2015090776-appb-000003
如本文所用，术语“肽集合”所述的肽集合由选自下组的肽或多肽构成：至少两种选自本发明短肽或其衍生多肽。
优选地，本发明所述的肽集合中至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自本发明第二方面所述的短肽或其衍生多肽；更优选地，所述肽集合至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自SEQ ID NO.:1-15的短肽或其衍生多肽。此外，所述的肽集合还包括可以除SEQ ID NO.:1-15以外的分离的HPV表位肽，优选地，为HPV 16型E6-7蛋白表位肽。
在一中优选方式中，所述的肽集合中至少含有SEQ ID NO.:5和/或SEQ ID NO.:14所示的短肽，优选地，所述的肽集合还可以由除上述两种多肽以外，1、2、3、4、5、6、7、8、9、10、11、12或13种选自SEQ ID NO.:1-15的序列组成。
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肽是没有分离纯化的，但同样的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，“分离的肽”是指本发明多肽基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化本发明多肽。基本上纯化的多肽(融合蛋白)在非还原聚丙烯酰胺凝胶上能产生单一的主带。在本发明中，本发明多肽包括符合式I结构式的短肽或含有核心序列X的多肽。
本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。
一旦鉴定获得了相关的肽序列，就可以用重组法来大批量地获得相关肽序列。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相关肽(融合蛋白)。
此外，还可用化学方法直接合成相关肽序列片段。
人类乳头状瘤病毒(human papillomavirus,HPV)
人类乳头状瘤病毒(human papillomavirus,HPV)是一组环状DNA病毒的总称。目前发现HPV型别已超过150多种，其中超过40种型别可感染女性生殖道上皮，而与宫颈癌以及宫颈上皮内高度病变发生密切相关的高危型HPV涉及HPV16、18、26、31、33、35、39、45、52、53、56、58、66、67、69、73、82和97等20多种型别，其中，HPV16流行率在世界各国排第一。即便于国内各地区，在发现流行率高的HPV16，52和58型中HPV16也居首位。
现已清楚高危型HPV导致的子宫颈癌在妇女肿瘤发生中位居第三位。作为针对严重威胁妇女生殖健康的HPV病毒的预防和治疗措施，准确诊断不同高危型HPV 感染是关键的第一步。目前的诊断方法可主要分为三大类，即细胞学诊断、HPV DNA鉴定和HPV血清学检测。其中前两者已在临床中普遍应用，后者经济简便但目前仍然始终处于研究中。即，HPV血清学抗体检测虽然也用于基础和流行病学研究，但未能用于临床诊断实际。究其原因，就是相对其他病毒HPV免疫原性弱(感染后抗体应答水平低)，特别是它与其他病毒不同，存在6个月到二年内自我清除机制而至今不清楚抗体发生的时期，而目前普遍采用的E6、E7和L1诊断抗原或它们的抗原性肽又缺乏真正的特异性，乃至血清学方法检测准确率不高。
本发明所针对的HPV主要为HPV16型病毒，基因组序列GenBank No.:K02718.1。其中，致癌蛋白E6和E7的序列分别为SEQ ID NO.:16和SEQ ID NO.:17所示。
SEQ ID NO.:16
(MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL)
SEQ ID NO.:17
(MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP)
兔抗HPV16 E6-E7融合蛋白抗血清和精细表位鉴定
本发明所采用的HPV16型病毒可采用常规方法获得并进行测序、扩增或表达。一种优选的方法如下：依据HPV16原型病毒基因组序列(GenBank No.:K02718.1)，由上海捷瑞生物工程有限公司化学合成编码全长E6(158aa)和E7(98aa)蛋白串联表达基因(经DNA测序确证基因序列无误)，用常规分子克隆技术分别将它重组插入pRSET-A原核表达质粒。在用IPTG诱导带6x His标签的E6-E7融合蛋白后，通过SDS-PAGE电泳分析和使用6x His单抗的蛋白免疫印迹试验确认重组E6-E7蛋白高表达。进而使用电泳割胶回收方法，从IPTG诱导表达的宿主菌总蛋白中纯化重组E6-E7融合蛋白。最后，主动免疫4只新西兰白兔，加强二次免疫后获得用于两个靶蛋白线性表位扫描作图的兔抗HPV16 E6-E7融合蛋白抗血清。
先前本发明人已构建了专门用于抗原表位扫描作图的短肽生物合成的pXXGST-1融合表达载体(Xu et al.Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy.J Reprod Immunol,81:9-16,2009)。因此，首先运用DNA重组技术(经化学 合成编码DNA正负链片段，退火重组插入表达质粒，转化大肠杆菌BL21(DE3)宿主菌，重组克隆测序验证插入片段DNA序列，以及热诱导表达目的GST188-16聚肽融合蛋白等步骤)，表达了分别跨越全长HPV16型E6和E7蛋白序列相互重叠9个氨基酸残基的系列16聚肽融合蛋白。然后将表达的E6/P1-P19和E7/P1-P12短肽融合蛋白和电转移到0.22μm硝酸纤维素膜上，分别用兔抗重组E6和E7蛋白多抗进行表位扫描作图。结果在分别鉴定出9个(图1中P1-2、P5-6、P8、P12、P14、P17和P19)和7个(图4中P1-P6和P12)印迹反应阳性的16聚肽(融合蛋白)。
进一步构建表达了以GST188为载体的跨越HPV16型E6和E7蛋白上印迹反应性16聚的相互重叠7个残基的系列融合表达8肽(E6/P20～P94，E7/P13～P68)，继而用兔抗E6-E7多抗鉴定第一轮抗原性肽扫描作图显示的全部反应性肽中可能存在的表位及其抗体识别最小基序。结果根据各表位肽印迹反应阳性的多个8肽之间共同的氨基酸序列，在E6蛋白上确定了八个精细表位肽。它们是用单一字母表示的PQER12-15、LECVYCK35-41、REVYDF47-52、YRDGNPY61-67、LYGTTLEQ90-97、RCINC109-113、RFHNIRG131-137和ETQL155-158(E6-1表位位于P1和P2重叠区)；在E7蛋白上确定了七个表位肽，它们是TLHEYML7-13、MLDLQ12-17、DLYC21-24、DSSEE30-34、DEIDGP36-41、AGQAEP42-47(应为42-47)和SQKP95-98(E7-2和E7-6分别位于P1和P2以及P5和P6的重叠区)。
应用
本发明HPV16型15个最小基序BCE肽的鉴定，可以用于HPV的血清学诊断，并为今后设计研制治疗性HPV多表位肽疫苗(以诱发细胞免疫的T细胞表位为主)，以及制备用于基础研究的各表位抗体奠定了基础。
本发明HPV最小基序表位肽的最重要用途是，它们或单独或组合被化学合成，或者与GST蛋白融合表达形成融合载体蛋白，可用作开发检测HPV16型病毒感染抗体生成的ELISA、肽芯片法和蛋白免疫印迹法检测用肽抗原，有助于建立可应用于临床诊断和流行病学研究的HPV16感染血清学检测方法。
此外，将本发明最小基序肽可以用作免疫原，将其施用于需要的对象后，可以刺激T细胞反应性，从而预防和/或治疗HPV感染。
HPV的检测或诊断
本发明提供了采用表位肽对HPV(尤其是16型)进行诊断的方法。这些试验是本领域所熟知的。试验中检测抗HPV蛋白抗体，可用于诊断HPV感染。
检测样品中是否存在抗HPV蛋白抗体的方法可利用本发明多肽与样品中抗HPV特异性结合的原理进行检测，它包括：通过样品与本发明多肽或其融合表达蛋白的抗原抗体反应，观察检测样品OD值是否大于对照二倍，或者观察是否形成抗体复合物(印迹条带)，OD值高或形成印迹条带就表示样品中存在抗HPV蛋白抗体或某一表位的抗体。
可以将本发明表位肽(或肽集合)固定点样在蛋白质芯片上从而形成试剂或试剂盒，用于检测样品中的抗HPV蛋白抗体。
此外，可将本发明多肽(化学合成肽，或与载体蛋白偶联的融合蛋白，或通过构建重组多表位肽抗原)，点样于检测板或检测试纸条。在检测时，使待测样品与本发明多肽发生免疫反应，从而诊断待测样品中是否存在人HPV抗体和/或存在哪些人HPV表位的抗体。
在本发明中，检测板可采用本领域常用的检测板材料，采用常规的检测板制备方法制成。代表性的免疫检测板包括测试条和支撑测试条的支撑板，如可采用PVC聚脂胶板等；所述的测试条由滤纸、层析材料、硝酸纤维素膜和吸水纸依次搭接组成，搭接部位可以采用常规的方法，如胶带等固定连接；其中：层析材料预包被胶体金或有色标记的本发明多肽或肽集合，优选地与偶联有载体蛋白的本发明的肽集合，且层析材料预包被有胶体金，硝酸纤维素膜上吸附检测线和质控线。
药物组合物和给药方式
本发明还提供了一种药物组合物。本发明的药物组合物可以是治疗性的或预防性的(如疫苗)。本发明的药物组合物包括有效量的本发明的表位肽或肽集合、或用该短肽致敏的树突状细胞或用树突状细胞诱导的T细胞，以及至少一种药学上可接受的载体、稀释剂或赋形剂。
在本发明中，这些(疫苗)组合物包含免疫性抗原(包括本发明短肽、肽集合或其衍生物)，并且通常与“药学上可接受的载体”组合，这些载体包括本身不诱导产生对接受该组合物的个体有害的抗体的任何载体。合适的载体的例子包括(但并不限于)蛋白质、脂质凝集物(如油滴或脂质体)等。这些载体是本领域普通技术人员所熟知的。另外，这些载体可起免疫刺激剂(“佐剂”)作用。
此外，本发明的(疫苗)组合物还可含有额外的佐剂。代表性的疫苗佐剂包括(但 并不限于)以下种类：无机佐剂，如氢氧化铝，明矾等；合成佐剂，如人工合成的双链多聚核苷酸(双链多聚腺苷酸、尿苷酸)、左旋咪唑、异丙肌苷等；油剂，如弗氏佐剂、花生油乳化佐剂、矿物油、植物油等。
通常，可将疫苗组合物或免疫原性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。该制剂还可乳化或包封在脂质体中，以增强佐剂效果。
组合物可制成单元或多元剂型。各剂型包含为了产生所期望的治疗效应而计算出预定量的活性物质，以及合适的药剂学赋形剂。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉内、瘤内、肌内、腹膜内、皮下、皮内、癌旁、或局部给药。
使用(疫苗)组合物时，是将安全有效量的本发明表位肽或肽集合施用于人，其中该安全有效量通常至少约1微克肽/千克体重，而且在大多数情况下不超过约8毫克肽/千克体重，较佳地该剂量是约1微克-1毫克肽/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明有益效果
本发明鉴定了HPV 16型E6和E7蛋白上的一组最小基序表位肽，可特异性地与抗人HPV抗体结合。本发明构建了真正特异的检测抗原，通过检测暴露在靶蛋白上多个表位诱发的抗体而提高临床检测的灵敏度，具体体现在可增加检测血清样本的稀释度，由此可减少体内众多低水平抗不明抗原抗体的干扰(随着稀释度增加而降低它们的检测灵敏度)，极大地降低或完全避免假阳性检测结果。此外，利用本发明最小基序肽组成的疫苗组合物，可有望用于主动免疫高危对象，通过刺激T细胞产生免疫应答，并达到预防和/或治疗HPV的作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：HPV16 E6和E7蛋白第一轮抗原性肽扫描作图
材料和方法：
1.HPV16型E6和E7蛋白串联编码基因根据原型HPV16基因组序列(GenBank No.:K02718.1)，由上海捷瑞生物工程有限公司化学合成。原核表达质粒pBV21由中国科学院遗传学和发育生物学研究所孙方臻教授惠赠，pRSET C质粒购自美国Invitrogen公司。热诱导表达质粒pXXGST-1和pXXGST-2由本专利发明人构建(专利号：ZL 200710173305.2)。大肠杆菌BL21(DE3)菌株购自北京康为世纪生物科技有限公司。
2.限制性内切酶EcoR I、BamH I、Sal I、Taq酶和T4DNA连接酶购自日本TaKaRa Biotechnology公司，预染蛋白分子量标准、辣根过氧化酶标记的羊抗兔二抗(IgG/HRP)、二氨基联苯胺(DAB)和0.2μm硝酸纤维素膜购自由上海生工生物技术服务公司代理的德国公司(Whatman GmbH,Dossel,Germany)。6×His单抗购自美国Aogma公司(货号：9618)。
3.QIAprep spin miniprep Kit质粒抽提试剂盒、QIAquick PCR产物纯化试剂盒和quick凝胶回收试剂盒购自德国QIAGEN公司。
4.新西兰白兔购自上海BK实验动物有限公司。EcoR I和Sal I以及BamH I和EcoR I
5.两端分别为BamH I和Sal I粘性末端，中间为各8/16肽编码DNA序列加TAA终止密码子的正负链DNA片段由上海捷瑞生物工程有限公司合成。
6.抗HPV16 E6-E7融合蛋白抗血清制备参照文献方法(宋力雯等.人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子-半透明带结合.生理学报，57：682-688,2005)。制备过程摘要如下：1)通过IPTG诱导方式表达重组HPV16 E6-E7融合蛋白；2)用制备胶电泳方法分别纯化两个目的蛋白；3)用纯化的重组E6-E7融合蛋白作为免疫原，经弗氏佐剂乳化后主动免疫4只雄性新西兰白兔；4)加强免疫二次后抽取血清，储存-20℃冰箱备用。
HPV16 E6和E7蛋白第一轮抗原性肽作图的具体步骤如下：
1.依据HPV16型E6和E7基因序列公开信息，分别设计跨越全长E6蛋白(aa 1-158)和E7蛋白(aa 1-98)序列相互重叠9个氨基酸残基的系列16肽编码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片段合成。
2.将1或2个OD的互补正负链片段用ddH2O溶解成20μmol/μl储存液(根据DNA合成报告单数据)；各取10μl储存液和20ddH2O于1.5ml Eppendorf管中，94℃水浴加热5min,自然降至室温后，在15μl反应体积中吸入2μl退火片段、1μl 约200ng/μl经BamH I和Sal I双酶切的pXXGST-1质粒、1μl T4 DNA连接酶和其1.5μl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℃培养过夜；第二天挑取氨苄LB平板上长出的单克隆转接3ml 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GST188-16肽(E6/P1-P19和E7/P1-P12)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2kDa)，挑取各重组短肽克隆进行DNA测序；
3.将插入片段测序结果正确的重组E6和E7短肽克隆接种加入Amp的3ml LB培养液中，于30℃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℃振荡培养2～3h至菌体浓度达到0.6～0.8OD后，调高温度至42℃热诱导4h，离心收集菌体，加上样裂解液煮沸5min备用；关于E6-E7克隆的表达，则在37℃培养过夜，翌日晨按1/50比例转接新鲜含Amp的LB培养液中，37℃振荡培养后加IPTG诱导4h。
4.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(100mA)转移2h，用丽春红染膜2min，在目的16聚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；
5.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8～10ml反应液中加入30μl一抗兔抗重组E6-E7融合蛋白血清或正兔血清，室温反应2h，PBS缓冲液洗涤后加入5μl二抗羊抗兔IgG/HRP(1:10000稀释)，室温反应1h，用PBS缓冲液洗涤后用ECL化学发光试剂盒显色(图1和图2)。
6.依据用抗E6-E7融合蛋白抗血清的印迹结果(图1和图2)，确定HPV16型E6和E7蛋白上分别存在九个(P1-2、P5-6、P8、P12、P14、P17和P19)和七个(P1-P6和P12)反应性16聚肽。
实施例2：HPV16 E6蛋白第二轮两个代表性抗原性肽中表位最小基序的鉴定
材料和方法：
见实施例1相应部分。
E6-2和E6-3表位最小基序鉴定的具体步骤如下：
1.依据实施例1中第一轮E6蛋白16聚抗原性肽作图结果，以P5和P6反应性肽为例进行它们的抗体识别最小基序鉴定。设计跨越P5和P6全长序列相互重叠7个氨基酸残基的系列8肽编码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片段合成。
2-4操作步骤同实施例1中的2-4步骤。
5.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8～10ml反应液中加入30μl一抗兔抗人重组E6-E7蛋白血清(1:2000稀释)，室温反应2h，PBS缓冲液洗涤后加入5μl二抗羊抗人IgG/HRP(1:10000稀释)，室温反应1h，用PBS缓冲液洗涤后进行ECL化学发光显色(图3A和图4A)。
6.依据它们中连续3个(图3A中Lanes 1-2，E6/P37-P38)和4个(图4A中Lanes 4-6，E6/P47-P49)印迹阳性片段共有残基序列(图3B和图4B)，确定两个表位最小基序分别为LECVYCK和REVYDF。
实施例3：HPV16 E7蛋白第二轮两个代表性抗原性肽中表位最小基序的鉴定
材料和方法：
见实施例1相应部分。
E7-3和E7-4表位最小基序鉴定的具体步骤如下：
1.依据实施例1中第一轮E7蛋白16聚抗原性肽作图结果，以P3和P4反应性肽为例进行它们的抗体识别最小基序鉴定。设计跨越P3和P5全长序列相互重叠7个氨基酸残基的系列8肽编码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片段合成。
2-4操作步骤同实施例1中的2-4步骤。
5.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8～10ml反应液中加入30μl一抗兔抗人重组E6-E7蛋白血清(1:2000稀释)，室温反应2h，PBS缓冲液洗涤后加入5μl二抗羊抗人IgG/HRP(1:2000稀释)，室温反应1h，用PBS缓冲液洗涤后进行ECL化学发光显色(图5A和图6A)。
6.依据它们中连续2个(图5A中Lanes 1-5，E7/P29-P33)和4个(图6A中Lanes 2-5，E7/P39-P42)印迹阳性片段共有残基序列(图5B和图6B)，确定两个表位最小基序分别为DLYC和DSSEE。
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。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种分离的肽集合，其特征在于，所述的肽集合由选自下组的肽或多肽构成：至少两种选自式I所述的短肽或其衍生多肽,其中
    X1-X-X2
    式I，
    并满足以下特征：
    (a)式I中X为4-8个氨基酸的核心片段；较佳地为4、5、6、7、或8个氨基酸；
    (b)式I中X1、X2为无、1、2或3个氨基酸，且X1和X2氨基酸个数总和≤4，较佳地，≤3、2、1，更佳地为0；
    (c)所述的核心片段的序列选自SEQ ID NO.:1-15中的一个或多个，较佳地为1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15个；和
    (d)所述的短肽具有与抗HPV16型E6-E7蛋白抗体结合的活性；
    其中，“-”表示肽键或肽接头。
  2. 如权利要求1所述的肽集合，其特征在于，所述的肽集合中至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自式I所述的短肽或其衍生多肽，优选地，所述肽集合至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自SEQ ID NO.:1-15的短肽或其衍生多肽。
  3. 如权利要求1所述的肽集合，其特征在于，所述的肽集合还包括除SEQ ID NO.:1-15以外的分离的HPV表位肽，优选地，为HPV 16型E6-7蛋白表位肽。
  4. 如权利要求1所述的肽集合，其特征在于，所述的肽集合中至少含有SEQ ID NO.:5和/或SEQ ID NO.:14所示的短肽。
  5. 一种分离的短肽，所述短肽的结构如式I所示：
    X1-X-X2
    式I，
    并满足以下特征：
    (a)式I中X为4-8个氨基酸的核心片段；较佳地为4、5、6、7、或8个氨基酸；
    (b)式I中X1、X2为无、1、2或3个氨基酸，且X1和X2氨基酸个数总和≤4，较佳地，≤3、2、1，更佳地为0；
    (c)所述的核心片段的序列选自SEQ ID NO.:1-15中的一个或多个，较佳地为1、2、3、4、5、6、7、8、9、10、11、12、13、14、或15个；和
    (d)所述的短肽具有与抗HPV16型E6-E7蛋白抗体结合的活性；
    其中，“-”表示肽键或肽接头。
  6. 如权利要求5所述的短肽，其特征在于，所述的短肽选自如SEQ ID NO.:1-15所示的短肽。
  7. 一种表达载体，其特征在于，所述的表达载体表达权利要求5所述的短肽或权利要求1所述的肽集合。
  8. 权利要求5所述短肽、或权利要求1所述的肽集合的用途，其特征在于，用于制备诊断或检测人乳头瘤病毒(HPV)的试剂和试剂盒。
  9. 一种试剂盒，其特征在于，所述的试剂盒含有特异性检测权利要求5所述短肽、或权利要求1所述肽集合的检测试剂，和说明书。
  10. 权利要求5所述短肽或权利要求1所述肽集合的用途，其特征在于，(a)用于制备预防和/或治疗人乳头瘤病毒的药物组合物；(b)作为抗原用于制备检测试剂；和/或(c)作为免疫原用于制备治疗药物。
  11. 一种药物组合物，其特征在于，所述组合物含有权利要求5所述短肽或权利要求1所述肽集合，和药学上可接受的载体。
  12. 一种体外非诊断性确定样本是否含有人乳头瘤病毒HPV的方法，其特征在于，检测样本中权利要求5所述短肽或权利要求1所述肽集合的表达量E1，并与正常样本中权利要求5所述短肽或权利要求1所述肽集合的表达量E0进行比较，若E1≥2E0，则说明该样本中含有人乳头瘤病毒HPV。
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