WO2016090540A1 - Minimal motif peptide of antigen epitope in human papilloma virus (hpv) 18-type cancerigenic e6 and e7 protein - Google Patents

Abstract

The present invention provides a minimal motif peptide of antigen epitope in human papilloma virus (HPV) 18-type cancerigenic E6 and E7 protein. The peptide can be specifically bound with an HPV18-type antibody and can be used in combination. The peptide can be applied to prevent, treat and detect HPV18-type virus infection.

Description

Epitope minimal motif peptide of human papillomavirus (HPV) type 18 oncogenic E6 and E7 proteins Technical field

The present invention is in the field of biomedicine, and in particular, the present invention relates to an epitope minimal motif peptide of the human papillomavirus (HPV) type 18 oncogenic E6 and E7 proteins.

Background technique

Since a B cell epitope (BCE, or antigenic determinant) capable of inducing the production of a linear or non-conformed antibody can be found or used as a candidate peptide for the development of an effective prophylactic synthetic peptide vaccine of an antibody, It is commonly used in the fields of immunology, virology and translational medicine, using mature conventional chemical synthetic peptide methods, phage display library methods, different long and short peptide chip methods, and computer software to predict and identify BCE peptides on target proteins of known amino acid sequences. An important research task.

It is now clear that the BCE peptide identified with the rabbit polyclonal antibody (polyclonal antibody) consists of 3-8 amino acid residues. However, due to the limitation of the epitope scanning mapping method, the peptides currently available for the synthesis of peptide vaccines and clinical diagnosis are longer, that is, they are actually an antigenic peptide containing the target epitope of the target antigen. Therefore, it cannot be truly called a BCE peptide. The drawbacks of such antigenic peptides in practical applications are obvious, that is, the longer the peptide, the more N potential epitopes therein. Therefore, as a candidate epitope peptide of a synthetic peptide vaccine, the effect of a specific functional/protective antibody is induced by the free parent protein antigen alone, and the uncertainty or effect is not optimal (because N in this antigenic peptide) Among the epitopes, which potential epitope is the dominant epitope of the induced antibody cannot be known or determined). As a diagnostic peptide antigen, there is often a problem of lack of specificity, especially in the case of autoimmune diseases and low titers of natural infection of the virus, since the dilution of the test serum sample is mostly impossible (common 1:10-1: 100), plus the fact that there are thousands of anti-unknown antigen antibodies in the human body, which may lead to higher or a certain ratio of false positive test results, and most tests do not allow false positive results, because that will cause Unacceptable and unnecessary harm to normal people without medication.

Therefore, from the practical application of epitope peptides, it is particularly important to identify the minimal recognition of antibodies for each target protein epitope. The necessity is at least two aspects: 1) potential N number of epitopes in the identified epitope peptide (for example, taking the 4 peptide epitope as an example, it may also have two other potential epitopes, ie The composition of 1-3 and 2-4 amino acid sequences is minimized, thereby greatly increasing the probability of inducing the antibody of interest, or reducing the false positive misdiagnosis rate of the antibody of interest; 2) revealing all linear epitopes on the target protein antigen, for example, Only one long antigenic peptide was identified by the rabbit polyclonal antibody against epitope mapping of HPV18-E7 protein (Bleul C, et al. Human papillomavirus type 18E6 and E7antibodies in human sera:increased anti-E7prevalence in cervical cancer patients.J Clin Microbiol, 29: 1579-1588, 1991), is due to the inability to identify each of the antibody-recognizing minimal motifs for many adjacent immunoblot reactive antigenic peptides (see Figures 2 and 3 of the specification). The latter can decode the benefits of all the fine epitope peptides on a target protein, and it is possible to combine them to construct a truly specific detection antigen, and to improve the sensitivity of clinical detection by detecting antibodies induced by multiple epitopes on the target protein. It can increase the dilution of the test serum samples, thereby reducing the interference of many low levels of anti-anti-antigen antibodies, greatly reducing or completely avoiding false positive test results).

Based on the above-mentioned concepts and practical application requirements, the inventors established an improved biosynthetic peptide method for epitope scanning mapping and epitope minimization identification (Xu WX, et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4using a peptide biosynthesis strategy.J Reprod Immunol,81:9-16,2009;Xu WX, Et al. Mapping of minimal motifs of B-cell epitopes on human zona pellucida glycoprotein-3. Clin Dev Immunol, 2012, 831010), and conduct related research, such as HPV58-, the earliest application and authorized antibody-based recognition minimal motif. L1 protein 16/18 epitope peptide invention patent (Xu Wanxiang et al. Linear epitope epitope minimal peptide of human papillomavirus type 58 L1 protein and its application. Patent No.: CN 101962400B; Xu WX, et al. Minimal motifs Of linear B-cell epitopes in L1 protein from human papillomavirus type 58 and their applications. Patent No.: US 8,715,681 B2). Obviously, based on the advantages and application prospects of fine epitope peptides, it is an invention to find an antigenic peptide (sometimes also mistakenly called an epitope peptide) from a known sequence target protein, from a target protein and / The fine epitope peptides identified therein or one of its antigenic peptides are also more important and practical applications.

Human papillomavirus (HPV) is a generic term for a group of circular DNA viruses. At present, more than 150 types of HPV have been found, of which more than 40 types can infect female genital tract epithelium, and high-risk HPV which is closely related to cervical cancer and cervical intraepithelial lesions involves HPV16, 18, 26, 31, More than 20 types, such as 33, 35, 39, 45, 52, 53, 56, 58, 66, 67, 69, 73, 82, and 97. In China, high-risk HPVs with high prevalence are types 16, 18, 52 and 58 (Shi JF, et al. Epidemiology and prevention of human papillomavirus and cervical cancer in China and Mongolia. Vaccine, 26: M53–M59, 2008; Jiang Z, et al. Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris. Protein Expr Purif, 80: 203-210, 2011).

As a preventive and therapeutic measure for the high-risk oncogenic HPV virus, which is a serious threat to women's reproductive health, accurate diagnosis of different high-risk HPV infections is a key first step. Current diagnostic methods can be broadly divided into three major categories, namely cytological diagnosis, HPV DNA identification, and HPV serological testing. The former two have been applied in the clinic. The latter is economically simple and has been the technology that has been studied, but it has not been used for clinical diagnosis. It is currently only used in basic and epidemiological studies. The reason is mainly due to the lack of true specificity of the currently used diagnostic antigens (although from the protein level, the E6, E7, L1 and L1-VLP pseudoviral proteins of each HPV type can be considered to be specific), as well as humans. In vivo, the level of antibody to HPV-encoded protein is low, and the detection accuracy is not high.

In view of the above-mentioned technical development background, there is an urgent need in the art to develop a novel high specificity and high sensitivity recombinant multi-epitope peptide detection antigen.

Summary of the invention

It is an object of the present invention to provide an epitope minimal motif peptide of the human papillomavirus (HPV) type 18 oncogenic E6 and E7 proteins.

In a first aspect of the invention, a minimal epitope motif peptide of a human papillomavirus type 18 E6 protein having the amino acid sequence of SEQ. ID No. 1 and designated HPV18E6 is provided. ^-16-10; or an amino acid sequence shown in SEQ.ID No. 2, referred to as HPV18E6-2 ^76-81; or having SEQ.ID No.3 amino acid sequence, referred to as HPV58E6-3 ^113-121; Or has the amino acid sequence shown in SEQ. ID No. 4, which is designated as HPV58E6-4 ^133-137 ; or has the amino acid sequence shown in SEQ. ID No. 5, and is referred to as HPV58E6-5 ^154-158 .

According to a second aspect of the present invention, a minimal epitope motif peptide of human papillomavirus type 18 E7 protein having the amino acid sequence of SEQ. ID No. 6 and designated as HPV18 is provided. E7-1 ^10-13 ; or has the amino acid sequence shown in SEQ. ID No. 7, which is designated as HPV18E7-2 ^20-26 ; or has the amino acid sequence shown in SEQ. ID No. 8, and is referred to as HPV18E7-3 ^30-35 Or has the amino acid sequence shown in SEQ.ID No. 9 and is referred to as HPV18E7-4 ^38-42 ; or has the amino acid sequence shown in SEQ.ID No. 10, which is designated as HPV18E7-5 ^46-49 ; or has SEQ.ID The amino acid sequence shown in No. 11 is referred to as HPV18E7-6 ^101-105 .

In a third aspect of the invention, a short peptide comprising the minimal epitope motif peptide of claim 1 is provided, the short peptide:

An amino acid sequence represented by X ₁ X ₂ DPTRRX ₃ , designated HPV18E6-1-1 (preferably as SEQ. ID No. 12), wherein X ₁ , X ₂ , and X ₃ are independently non-independent Specific amino acid residues are either none; or

An amino acid sequence represented by X ₁ RELRHYX ₂ , designated HPV18E6-2-1 (preferably as SEQ. ID No. 13), wherein X ₁ and X ₂ are independently non-specific amino acid residues or No; or

Having the amino acid sequence of X ₁ NPAEKLRHLX ₂ , designated HPV18E6-3-1 (preferably as shown in SEQ. ID No. 14), wherein X ₁ and X ₂ are independently non-specific amino acid residues or No; or

Having the amino acid sequence of X ₁ X ₂ HYRGQX ₃ , designated HPV18E6-4-1 (preferably as SEQ. ID No. 15), wherein X ₁ , X ₂ , and X ₃ are independently non-independent Specific amino acid residues are either none; or

An amino acid sequence represented by X ₁ X ₂ X ₃ RETQV, designated HPV18E6-5-1 (preferably as SEQ. ID No. 16), wherein X ₁ , X ₂ , and X ₃ are each independently Non-specific amino acid residues are either absent or not.

A fourth aspect of the invention provides a short peptide comprising a minimal epitope motif peptide according to the second aspect of the invention, wherein the short peptide has:

Having the amino acid sequence of X ₁ X ₂ DIVLX ₃ X ₄ , designated HPV18E7-1-1 (preferably as SEQ. ID No. 17), in the sequence of X ₁ , X ₂ , X ₃ , and X ₄ Individually independent of non-specific amino acid residues or none; or

Having the amino acid sequence of X ₁ EIPVDLLX ₂ , designated HPV18E7-2-1 (preferably as SEQ. ID No. 18), wherein X ₁ and X ₂ are independently non-specific amino acid residues or No; or

An amino acid sequence represented by X ₁ QLSDSEX ₂ , designated HPV18E7-3-1 (preferably as SEQ. ID No. 19), wherein X ₁ and X ₂ are independently non-specific amino acid residues or No; or

Having the amino acid sequence of X ₁ X ₂ NDEIDX ₃ , designated HPV18E7-4-1 (preferably as shown in SEQ. ID No. 20), wherein X ₁ , X ₂ , and X ₃ are independently non-independent Specific amino acid residues are either none; or

Having the amino acid sequence of X ₁ X ₂ HQHLX ₃ X ₄ , designated HPV18E7-5-1 (preferably as shown in SEQ. ID No. 21), in the sequence of X ₁ , X ₂ , X ₃ , and X ₄ Individually independent of non-specific amino acid residues or none; or

An amino acid sequence represented by X ₁ X ₂ X ₃ CASQQ, designated HPV18E7-6-1 (preferably as SEQ. ID No. 22), wherein X ₁ , X ₂ , and X ₃ are independently Non-specific amino acid residues are either absent or not.

According to a fifth aspect of the present invention, there is provided an epitope minimization peptide of human papillomavirus (HPV) type 18, which is derived from E6 or E7 protein of papillomavirus type 18, and The length of the motif peptide is ≤9 amino acids, and the motif peptide has an activity of binding to an anti-HPV18 type antibody.

In another preferred embodiment, the motif peptide may be 4, 5, 6, 7, 8, 9 amino acids in length.

In another preferred embodiment, the ability of the short peptide formed after removal of any one of the amino acids of the motif peptide to bind to the antibody is significantly reduced (preferably, the binding ability is reduced by more than 30%; more preferably, the binding ability is decreased by 50%) Above, most preferably the binding capacity is reduced by more than 90%) or lost.

In another preferred embodiment, the antibody comprises a monoclonal antibody, a polyclonal antibody or an antiserum.

In another preferred embodiment, the motif peptide is derived from an E6 protein, and the motif peptide is selected from the group consisting of:

DPTRR (SEQ ID NO.: 1), RELRHY (SEQ ID NO.: 2), NPAEKLRHL (SEQ ID NO.: 3), HYRGQ (SEQ ID NO.: 4), and RETQV (SEQ ID NO.: 5).

In another preferred embodiment, the motif peptide is derived from an E7 protein, and the motif peptide is selected from the group consisting of:

DIVL (SEQ ID NO.: 6), EIPVDLL (SEQ ID NO.: 7), QLSDSE (SEQ ID NO.: 8), NDEID (SEQ ID NO.: 9), HQHL (SEQ ID NO.: 10) and CASQQ (SEQ ID NO.: 11).

In another preferred embodiment, the E6 protein comprises wild-type and mutant E6 proteins.

In another preferred embodiment, the E7 protein comprises a wild type and a mutant E7 protein.

According to a sixth aspect of the invention, there is provided an antigenic peptide of human papillomavirus (HPV) type 18, said antigen peptide structure being as shown in formula I:

Ya-Y-Yb, I,

And meet the following characteristics:

(a) In the formula I, Y is the motif peptide of claim 5;

(b) In the formula I, Ya and Yb are independently a fragment consisting of no 1, 2, or 3 amino acids, and the total number of Y1 and Y2 amino acids is ≤4;

(c) the antigen peptide has an activity of binding to an anti-HPV antibody;

Wherein "-" means a peptide bond or a peptide linker.

In another preferred embodiment, the antigenic peptide sequence is identical to the specific sequence of the native HPV type 18 E6 or E7 protein (the specific sequence refers to a truncated fragment derived from the HPV type 18 E6 or E7 protein).

In another preferred embodiment, the motif peptide is derived from an E6 protein, and the motif peptide is selected from the group consisting of:

DPTRR, RELRHY, NPAEKLRHL, HYRGQ and RETQV.

In another preferred embodiment, the antigenic peptide is selected from the group consisting of:

Y ₁ Y ₂ DPTRRY ₃ , Y ₁ RELRHYY ₂ , Y ₁ NPAEKLRHLY ₂ , Y ₁ Y ₂ HYRGQY ₃ , and Y ₁ Y ₂ Y ₃ RETQV, wherein each Y ₁ , Y ₂ , and Y ₃ represents an amino acid residue, respectively .

In another preferred embodiment, the antigenic peptide is selected from the group consisting of:

FEDPTRRP, IRERRHYS, HNPAEKLRHLN, AGHYRGQC, and QRRRETQV.

In another preferred embodiment, the motif peptide is derived from an E7 protein, and the motif peptide is selected from the group consisting of:

DIVL, EIPVDLL, QLSDSE, NDEID, HQHL and CASQQ.

In another preferred embodiment, the antigenic peptide is selected from the group consisting of:

Y ₁ Y ₂ DIVLY ₃ Y ₄ , Y ₁ EIPVDLLY ₂ , Y ₁ QLSDSEY ₂ , Y ₁ Y ₂ NDEIDY ₃ , Y ₁ Y ₂ HQHLY ₃ Y ₄ , and Y ₁ Y ₂ Y ₃ CASQQ, wherein each Y ₁ , Y ₂ , Y ₃ , and Y ₄ represent an amino acid residue, respectively.

In another preferred embodiment, the antigenic peptide is selected from the group consisting of:

LQDIVLHL, NEIPVDLLC, EQLSDSEE, EENDEIDG, VNHQHLPA, and CPWCASQQ.

In another preferred embodiment, the antigenic peptide is obtained by chemical synthesis or bioengineering methods, including recombinant proteins, fusion proteins.

According to a seventh aspect of the invention, a fusion protein comprising the motif peptide of the fifth aspect of the invention or the antigen peptide of the sixth aspect of the invention, and a vector derived from the non-HPV protein Sequence, and optional tag sequences.

In an eighth aspect of the invention, there is provided an isolated polypeptide collection, the peptide collection consisting of two or more polypeptides selected from the group consisting of one or more of the following:

(i) one or more motif peptides selected from the fifth aspect of the invention; and/or

(ii) one or more antigenic peptides selected from the sixth aspect of the invention; and/or

(iii) one or more fusion proteins selected from the seventh aspect of the invention.

The ninth aspect of the invention provides the motif peptide according to the first or fifth aspect of the invention, the antigen peptide of the sixth aspect of the invention, the fusion protein of the seventh aspect of the invention, or the eighth aspect of the invention Use of the polypeptide set of the aspect, for the preparation of a reagent or kit for detecting an HPV18 type E6 and/or E7 early protein-inducing antibody; or

For the preparation of a medicament for the prevention or treatment of HPV type 18 infection; or

For the preparation of a medicament for preventing or treating a disease caused by HPV type 18 infection, such as cervical cancer; or

It is used to detect E6 and/or E7 protein antibodies against HPV18.

In another preferred embodiment, the reagent comprises a test strip, a test plate, a protein chip, and a magnetic bead.

In another preferred embodiment, the anti-human papillomavirus antibody is an anti-human papillomavirus antibody in a human body fluid sample. Preferably, the body fluid is blood or saliva.

According to a tenth aspect of the invention, there is provided an immunoassay kit comprising the motif peptide of the first and fifth aspects of the invention; and/or the antigen peptide of the sixth aspect of the invention; / or the fusion protein of the seventh aspect of the invention. The kit may include one or more of the motif peptides of the first and fifth aspects of the invention, the antigenic peptide of the sixth aspect of the invention, and/or the fusion protein of the seventh aspect of the invention.

In another preferred embodiment, the kit further comprises a vector for detecting the motif peptide of the first and fifth aspects of the invention against the human papillomavirus antibody; the antigen peptide of the sixth aspect of the invention; And/or the fusion protein of the seventh aspect of the invention is coated on the carrier.

According to an eleventh aspect of the present invention, a method for preventing or treating a disease caused by HPV infection or HPV infection, which comprises administering to a subject in need thereof a safe and effective amount of the motif peptide of the first or fifth aspect of the present invention The antigen peptide according to the sixth aspect of the present invention, the fusion protein of the seventh aspect of the present invention, and the present invention The polypeptide assembly of the eighth aspect of the invention.

In another preferred embodiment, the HPV is of the HPV 18 type.

A twelfth aspect of the invention provides a method for detecting an anti-HPV18 type antibody, comprising the steps of: the motif peptide of the fifth aspect of the invention, the antigen peptide of the sixth aspect of the invention, and/or The fusion protein of the seventh aspect of the invention is contacted with a sample containing an anti-HPV18 type antibody and detects the formation of an antigen-antibody complex.

In another preferred embodiment, the detection is for non-diagnostic purposes.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1. Immunoblot identification of the first round of antigenic peptide mapping of HPV18E6 protein

Note: The antigen is a group of 16-mer peptides (number: E6/P1-P19, corresponding to lanes 1-19) that overlap 9 residues across the full-length HPV18E6 protein sequence, and the primary antibody is a 1:3000 dilution of rabbit anti-recombinant. E6 protein antiserum. No reactive bands were generated with pre-immune rabbit control sera (results not shown). The arrows indicate immunoblot reactive 16 peptides (fusion proteins), respectively.

Figure 2 Immunoblotting identification of the first round of antigenic peptide mapping of HPV18E7 protein

Note: The antigen is a group of 16-mer peptides (number: E7/P1-P12, corresponding to lanes 1-12) that overlap 9 residues across the full-length HPV18E7 protein sequence, and the primary antibody is a 1:3000 dilution of rabbit anti-recombinant. E7 protein antiserum. No reactive bands were generated with pre-immune rabbit control sera (results not shown). The arrows indicate immunoblot reactive 16-polypeptides (fusion proteins), respectively.

Figure 3. Immunoblot identification of HPV18E6-1 epitope peptide

A, a group of 8-mer peptides derived from E6 protein-reactive 16-mer peptides overlapping each other with 7 residues (E6/P20-P28) were identified by immunoblotting; B, based on epitopes of consensus residues in the reactive 8 peptide Minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E6 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide. The left-to-right lane in Figure A is from Figure B. The peptides from top to bottom correspond.

Figure 4. Immunoblot identification of HPV18E6-2 epitope peptide

A, a set of 8-mer peptides (E6/P29-P37) covering a full-length E6/P10 reactive 16-mer peptide overlapping 7 residues are identified by immunoblotting; B, based on a table of shared residues in the reactive 8 peptide Bit minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E6 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide. The left-to-right lane in Figure A is from Figure B. The peptides from top to bottom correspond.

Figure 5. Immunoblot identification of HPV18E6-3 epitope peptide

A, a group of 10-mer peptides derived from E6 protein-reactive 16-mer peptides overlapping 9 residues (E6/P62-P69) were identified by immunoblotting; B, based on epitopes of consensus residues in the reactive 10 peptide Minimum motif analysis. The arrows indicate the bands reactive with rabbit anti-recombinant E6 antiserum, the shades indicate the common peptide sequence (minimum motif peptide) in the reactive peptide, and the left-to-right lanes in Figure A The peptides from top to bottom in Figure B correspond.

Figure 6. Immunoblot identification of HPV18E6-4 epitope peptide

A, a group of 8-polypeptides derived from the E6 protein that overlap each other with 7 residues (only the last E6/P78-P84) were identified by immunoblotting, including the use of the N-terminus from the reactive P23. One residue of short peptide (E6/P78-P84) was deleted one by one; B, based on the minimal motif analysis of the consensus residue sequence in the reactive 8 peptide. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E6 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide. The left-to-right lane in Figure A is from Figure B. The peptides from top to bottom correspond.

Figure 7. Immunoblot identification of HPV18E6-5 epitope peptides

A, a set of 8-mer peptides (E6/P89-P96) covering a full-length E6/P19 reactive 14-mer peptide overlapping with 7 residues are identified by immunoblotting; B, based on a table of shared residues in the reactive 8 peptide Bit minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E6 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide. The left-to-right lane in Figure A is from Figure B. The peptides from top to bottom correspond.

Figure 8. Immunoblot identification of HPV18E7-1 epitope peptide

A, a group of 8-mer peptides derived from E7 protein-reactive 16-mer peptides overlapping each other with 7 residues (E7/P13-P22) were identified by immunoblotting; B, based on the epitope of the consensus residue sequence in the reactive 8 peptide Minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E6 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide. The left-to-right lane in Figure A is from Figure B. The peptides from top to bottom correspond.

Figure 9 Immunoblotting identification of HPV18E7-2 epitope peptide

A, a set of 8-polypeptide (E7/P29-P37) covering a full-length E7/P3 reactive 16-mer peptide overlapping with 7 residues, identified by immunoblotting; B, based on a table of shared residues in the reactive 8 peptide Bit minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E7 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide, the left-to-right lane in Figure A and the Figure B The peptides from top to bottom correspond.

Figure 10 Immunoblot identification of HPV18E7-3 epitope peptide

A, a group of 8-mer peptides derived from E7 protein-reactive 16-mer peptides overlapping each other with 7 residues (E7/P38-P44) were identified by immunoblotting; B, based on epitopes of consensus residues in the reactive 8 peptide Minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E7 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide, the left-to-right lane in Figure A and the Figure B The peptides from top to bottom correspond.

Figure 11 Immunoblot identification of HPV18E7-4 epitope peptide

A, a set of 8-polypeptide (E7/P45-P53) covering a full-length E7/P5 reactive 16-mer peptide overlapping with 7 residues, identified by immunoblotting; B, based on a table of shared residues in the reactive 8 peptide Bit minimum motif analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E7 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide, the left-to-right lane in Figure A and the Figure B The peptides from top to bottom correspond.

Figure 12 Immunoblot identification of HPV18E7-5 epitope peptide

A, a group of 8-mer peptides derived from E7 protein-reactive 16-mer peptides overlapping each other with 7 residues (E7/P54-P61) were identified by immunoblotting; B, based on the epitope of the consensus residue sequence in the reactive 8 peptide Minimum motif analysis. The arrows indicate the reactive bands produced by the rabbit anti-recombinant E7 protein antiserum, respectively. The shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide, and the left-to-right lane in Figure A corresponds to the top-down peptide in Figure B.

Figure 13 Immunoblot identification of HPV18E7-6 epitope peptide

A, a set of 8-mer peptides derived from 7 residues of E7 protein overlapped with each other (E7/P67-P75) was identified by immunoblotting; B, based on the minimal base of the consensus residue sequence in the reactive 8 peptide Sequence analysis. The arrows indicate the reactive bands generated with the rabbit anti-recombinant E7 protein antiserum, respectively, and the shading indicates the common peptide sequence (minimum motif peptide) in the reactive peptide, the left-to-right lane in Figure A and the Figure B The peptides from top to bottom correspond.

detailed description

The present inventors have obtained extensive and intensive studies to obtain a population of human papillomavirus (HPV) type 18 oncogenic E6 and E7 protein epitope minimolecular peptides, which specifically bind to HPV18 type E6 or E7 protein The antibody binds. The present invention has been completed on this basis.

HPV 18

Human papillomavirus (HPV) type 18 is a highly carcinogenic virus whose E6 and E7 proteins expressed in host cells are oncogenic proteins.

In a preferred embodiment of the invention, the amino acid sequence of the HPV18 E6 protein is as follows:

MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (158aa) (SEQ ID NO.: 12).

In a preferred embodiment of the invention, the amino acid sequence of the HPV18E7 protein is as follows: MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (105aa) (SEQ ID NO.: 13).

Linear B cell epitope

In general, most proteins are antigenic, that is, induce linear (non-conformational) B cell epitopes and conformational epitope antibodies against specific antigens in mammals, or induce helper T cells or cytotoxic T Cells secrete specific cytokine responses. Relative to the detection of antigen and antibody effective vaccine development, the most research is the use of chemical synthetic peptide method and phage display library technology to identify the linear epitope of the amino acid sequence in the target protein.

Motif peptide

In the present invention, the inventors established "Xu WX, et al. Minimal motif mapping of a known epitope on human zona pellucida protein-1 using peptide biosynthesis strategy. J Reprod Immunol, 2009; 81: 9-16; Xu WX, et al.Xu Wan-xiang, He Yaping, Wang Jian, Tang Hai-ping, Shi Hui-juan, Sun Xiao-xi, Ji Chao-neng, Gu Shao-hua and Yi Xie, Mapping of minimal motifs of B -cell epitopes on human zona pellucida glycopotein-3. Clin Dev Immunol, 2012; 2012: 831010.) Previously, it was not possible to identify antibody recognition tables with less than or equal to 9 residues of polypeptide using anti-recombinant polyclonal antibody. The minimal motif (the smallest epitope peptide of the epitope), therefore, strictly speaking, a peptide that has not been identified by the minimal motif of the epitope cannot be called an epitope or an epitope peptide, and can only be called an "antigenic peptide". . In other words, in the target protein epitope scanning mapping, only peptide sequences identified by the minimal motif of the epitope can be referred to as "epitope", "epitope peptide" or "epitope minimal motif peptide".

In the present invention, "the epitope peptide of the present invention", "the motif peptide of the present invention", "the epitope minimum peptide of the present invention" are used interchangeably, and refer to a carcinogenic E6 or E7 protein derived from papillomavirus, And the motif peptide has a length of ≤ 9 amino acids, and is a general term for an epitope ligand peptide having an activity of binding to an anti-HPV antibody.

Preferred motif peptides in the present invention are shown in Tables 2 and 4, respectively.

Antigenic peptide

The terms "antigenic peptide", "polypeptide of the invention" are used interchangeably to refer to a polypeptide comprising a peptide of the invention that has activity in binding to an anti-HPV antibody. It will be understood that the term also includes derivatives of the polypeptides of the invention, which refer to sequences of the polypeptide of the invention which are added via 1-3 amino acids, 1-2 deletions and still contain the motif peptide, and which have anti-human HPV The antibody binds to the active polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.

Table 1

Initial residue	Representative substitution	Preferred substitution
			Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
			Asn(N)	Gln;His;Lys;Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro; Ala	Ala
			His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
			Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, 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.

As used herein, "isolated peptide" refers to a polypeptide of the invention that is substantially free of other proteins, lipids, carbohydrates, or other antigenic peptides (not identified by the minimal epitope of the epitope) that are naturally associated therewith. 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. In the present invention, 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.

Once the relevant peptide sequences have been identified, 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.

In addition, related peptide sequences can be directly synthesized by chemical methods.

Preferred antigenic peptides of the invention are shown in Tables 3 and 5.

application

In addition to being useful for the diagnosis of HPV infection, the polypeptide of the present invention can also be used as a pharmaceutical composition or vaccine composition for the preparation of a disease preventing or treating HPV infection or HPV infection.

The invention relates to a diagnostic test method for qualitatively and quantitatively detecting anti-HPV E6/E7 protein antibody levels. These tests are well known in the art.

The method for detecting the presence or absence of an anti-HPV E6/E7 protein antibody in a sample can be detected by the principle that the polypeptide of the present invention specifically binds to an anti-HPV E6/E7 protein antibody in a sample, which comprises: expressing a fusion protein with a polypeptide of the present invention or a sample thereof The antigen-antibody reaction of the protein, observe whether the OD value of the test sample is twice larger than the control, or observe whether an antibody complex (imprint strip) is formed, or observe the fluorescent chromogenic antigen antibody reaction by the peptide chip method, and the OD value is high, and the imprint strip is formed. Band or fluorescent coloration indicates the presence of an antibody against the HPV E6/E7 protein antibody or an epitope in the sample.

The epitope peptide of the present invention can be immobilized on a protein chip to form a reagent or kit for detecting an anti-HPV E6/E7 protein antibody in the sample.

Furthermore, the 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. At the time of detection, the test sample is immunologically reacted with the polypeptide of the present invention, thereby diagnosing whether the HPV E6/E7 protein antibody and/or the HPV E6/E7 protein epitope antibody are present in the sample to be tested.

In the present invention, 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 present invention also provides a vaccine composition useful as an HPV vaccine, which vaccine (composition) can be used to achieve anti-tumor (HPV-positive tumor, such as cervical cancer) vaccine development. Typically, the vaccine contains: (i) a polypeptide of the invention as an immunologically active ingredient, and (i) a pharmaceutically or immunologically acceptable carrier.

In the present invention, the term "containing" means that the various ingredients may be applied together or in the composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "contains."

In the present invention, these vaccines comprise an immunological antigen (including a polypeptide of the present invention or a derivative thereof), and Generally, in combination with a "pharmaceutically acceptable carrier," such carriers include any carrier which does not itself induce the production of antibodies which are detrimental to the individual receiving the composition. Examples of 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").

In the present invention, the vaccine can be administered to a subject in a desired manner.

The main content of the present invention is to reveal all linear BCEs of HPV18E6 and E7 early proteins by biosynthetic peptide technology and rabbit polyclonal antibody. Although epitope mapping studies of two similar proteins have been performed earlier, antigenic peptides are obtained. (Bleul C, et al. Human papillomavirus type 18E6 and E7 antibodies in human sera:increased anti-E7prevalence in cervical cancer patients. J Clin Microbiol, 29: 1579-1588, 1991) instead of the epitope minimal motif peptide.

The present invention relates to all linear B cell epitopes (BCEs, BCEs, or antigenic determinants) of high-risk human papillomavirus (HPV) 18 E6 and E7 oncoproteins closely related to cervical cancer in women and minimum Motif peptide. The invention further relates to the eleven antigenic epitope minimal sequence and the extended 8-11 peptide of the HPV18 type E6 and E7 proteins, which can be used as a therapeutic multi-valent HPV for the preparation of cervical cancer including cytotoxic T cell epitopes. Candidate epitope peptides for vaccines, and their chemically synthesized peptide-specific infection detection antigens, either alone or in combination as methods for ELISA and/or microscopy of HPV18 virus infection, or for protein immunization by recombinant antigen alone or in combination Blot detection.

The object of the present invention is to provide 11 BCE peptides which are first identified by the applicant on the human papillomavirus (HPV) type 18 E6 and E7 proteins, and the BCE-containing minimal groups which can be chemically synthesized or expressed in fusion with the GST carrier protein. The 8 peptide is longer or longer than the 8 peptide antigen. The former can be used alone or in combination as an antigenic peptide for the design and development of a therapeutic HPV multi-epitope vaccine, and the latter 8 peptide or fusion protein thereof can be used for screening for the detection antigen of anti-HPV18E6 and E7 antibodies in the serum of normal women or cervical cancer patients. .

The content of the present invention is further described in detail as follows:

1. According to the HPV18 prototype viral genome sequence (GenBank No.: X05015.1), the full-length E6 (158aa) and E7 (105aa) protein genes were chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd. (the DNA sequence was confirmed by DNA sequencing) ), they were separately inserted into pBV221 and pRSET-A prokaryotic expression plasmids by conventional molecular cloning techniques. After heat-induced expression of E6 and induction of E7 protein with IPTG, high expression of recombinant E6 and E7 proteins was confirmed by Western blotting analysis by SDS-PAGE electrophoresis and using their specific monoclonal antibodies. Furthermore, the electrophoresis tapping recovery method was used (Zou Yongshui, Xu Wanxiang et al. Preparation of recombinant human chorionic gonadotropin chimeric peptide 12 by polyacrylamide gel electrophoresis. Journal of Biochemistry and Biophysics, 34: 671-674, 2002), respectively The recombinant E6 and E7 proteins were purified by heat-induced expression of the total host protein. Finally, four New Zealand white rabbits were actively immunized, respectively, and secondary anti-HPV18E6 and E7 protein antisera were obtained after secondary immunization for their linear epitope scanning.

2. In the present application, the pXXGST-1 fusion expression vector (Xu et al. Minimal motif mapping of a known epitope on human zona pellucida protein) which was previously constructed by the applicant and used for antigen epitope scanning mapping is used. -4using a peptide biosynthesis strategy. J Reprod Immunol, 81:9-16, 2009). Firstly, DNA recombination technology (chemically synthesized DNA positive and negative strand fragments, annealed recombinant insert expression plasmid, transformed E. coli BL21 (DE3) host strain, recombinant clone sequencing verified insert DNA sequence, and heat-induced expression of GST188-16 Peptide fusion protein and the like), expressed across the full length E6 and E7 protein sequences are heavy A series of 16 polypeptide fusion proteins with 9 amino acid residues. The expressed E6/P1-P19 and E7/P1-P12 short peptide fusion proteins were then electrotransferred onto a 0.22 μm nitrocellulose membrane and subjected to epitope scanning mapping with rabbit anti-recombinant E6 and E7 protein polyclonal antibodies, respectively. As a result, nine (P1-2, P9-11, P15-17, and P19 in Fig. 1) and seven (P1-P6 and P12 in Fig. 2) were identified as positive 16 peptides (fusion proteins). .

3. Further constructing a series of fusion expression 8 peptides (E6/P20～P96, E7/P13～P75) expressing GST188 as a vector spanning the E6 and E7 proteins on the E6 and E7 proteins and overlapping 7 residues. The rabbit anti-E6 and E7 polyclonal antibodies were used to identify the epitopes that may be present in all reactive peptides displayed by the first round of antigenic peptide scanning mapping and their antibody recognition minimal motifs.

Results According to the common amino acid sequence between the 8 peptides positive for each epitope peptide imprinting, five epitope peptides were identified on the E6 protein, which were present in the reactive P1 and P2, P9 and P10, and P16 and P17 overlap, respectively. region, P15 and P19 minimal epitope peptide motif ^{DPTRR 6-10, RELRHY 76-81, NPAEKLRHL 113-121} , HYRGQ 133-137 and RETQV ^154-158; epitope peptides identified in six E7 proteins, they are Is DIVL ^10-13 , EIPVDLL ^20-26 , QLSDSE ^30-35 , NDEID ^38-42 , HQHL ^46-49 and CASQQ ^101-105

The identification of the 11 minimal motif BCE peptides of the above HPV18 E6 and E7 proteins laid the foundation for the design and development of therapeutic HPV multi-epitope peptide vaccines (mainly T cell epitopes for inducing cellular immunity). Another important use of these E6 and E7 minimal motif epitope peptides and their extended peptides (derived peptides) is that they are either chemically synthesized, either alone or in combination, or expressed in fusion with GST, and can be used as a development assay to detect HPV18 virus infection. Antigens for antibody-based ELISA, peptide microarray and Western blotting help to establish a serological assay for HPV infection that can be applied to clinical diagnosis.

The main advantages of the invention are:

(1) Firstly revealed a set of epitope-less minimal motif peptides of HPV18 type proteins, which specifically bind to anti-HPV18 type antibodies;

(2) The polypeptide of the present invention can be used in combination to identify a sample (especially a serum sample) because a plurality of antibodies exposed to multiple epitopes on the surface of the HPV type 18 E6 protein are detected (not usually detected by an antigenic peptide) Antibody) to diagnose HPV18 infection or HPV18 infection-causing diseases (cervical cancer, etc.); since single-epitope peptide antigens are highly specific and can be used in combination, the sensitivity of detection can be improved (increasing the dilution of serum samples) And specificity.

(3) The epitope minimal motif peptide of the present invention can also be used to prepare a vaccine composition against HPV infection, and any motif peptide minimizes the number of N potential epitopes that may be present, thereby maximizing the possibility of exclusion. It is a protein epitope of other tissues in the human body, or a non-HPV type 18 E6 protein specific epitope, and thus the application of the polypeptide of the present invention can avoid triggering a non-specific immune response against non-HPV proteins, and is more safe and effective.

The present invention will be further described in detail below with reference to specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. 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 the "Molecular Cloning: Laboratory Manual" by J. Sambrook and DW Russell. Society, 2002) and [United States] E. Harlow and D. Ryan compiled the steps described in the "Guide to the Experimental Guide to Antibody Technology" (Science Press, 2002), such as Shen Care, or according to the conditions recommended by the production vendor. . Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples can be used unless otherwise specified. Commercial channels are available.

Example 1: Linear mapping of the first round of antigenic peptides of human papillomavirus type 18 (HPV18) E6 and E7 proteins

Materials and Method:

1. HPV18E6 and E7 protein coding genes were chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd. according to the prototype HPV18 genome sequence (GenBank No.: X05015.1). The prokaryotic expression plasmid pBV221 was purchased from Youbio Biotechnology Co., Ltd., 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.: ZL200710173305.2). Escherichia coli BL21 (DE3) strain was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.

2. 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 μm nitrocellulose membrane were purchased from Shanghai Shenggong Biotechnology Service Company. 6×His monoclonal antibody was purchased from Shanghai Ruixing Biotechnology Co., Ltd.

3. QIAprep spin miniprep Kit Plasmid extraction kit, QIAquick PCR product purification kit and quick gel recovery kit were purchased from QIAGEN, Germany.

4. New Zealand white rabbits were purchased from Shanghai BK Laboratory Animals Co., Ltd. EcoR I and Sal I and BAMHI and EcoR I

5. Both ends of the BamH I and Sal I cohesive ends, and the positive and negative strand DNA fragments of the 8/16 peptide-encoding DNA sequence plus the TAA stop codon were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

6. Anti-HPV18E6 and E7 protein antiserum preparation reference literature method (Song Liwen et al. Immunogenicity of human egg zona pellucida ZP3a and ZP3b peptides and their antiserum inhibited human sperm-translucent band binding in vitro. Acta Physiologica Sinica, 57: 682-688, 2005). The preparation process is summarized as follows: 1) expression of recombinant HPV18E6 and E7 proteins by heat induction or IPTG induction; 2) purification of two target proteins by preparative gel electrophoresis; 3) purification of recombinant E6 and E7 proteins as immunogens, 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 specific steps of the first round of antigenic peptide mapping of HPV18E6 and E7 proteins are as follows:

1. Based on the disclosure information of HPV18 E6 and E7 gene sequences, design a series of 16 peptides encoding DNA positive and negative across the full length E6 protein (aa 1-158) and E7 protein (aa 1-105) sequences overlapping 9 amino acid residues. Chain fragment (5'-end plus 5'-gatcc, 3'-end plus taag-3'; negative 5'-end plus 5'-tcgactta, 3'-end plus g-3'), synthetic DNA Fragment.

2. Dissolve 1 or 2 OD complementary positive and negative strand fragments with ddH ₂ O into 20 μmol/μl stock solution (according to DNA synthesis report data); each 10 μl stock solution and 20 dd H ₂ O in 1.5 ml Eppendorf tubes, After heating in a 94 ° C water bath for 5 min, after naturally dropping to room temperature, 2 μl of the annealed fragment, 1 μl of about 200 ng/μl of the pXXGST-1 plasmid digested with BamH I and Sal I, 1 μl of T4 DNA ligase and 1.5 μl thereof were inhaled in a 15 μl reaction volume. The buffer was ligated overnight; the ligation solution was transformed into competent BL21 (DE3) host bacteria, coated on Amp-containing LB plates, and cultured overnight at 37 ° C; the next day, the monoclonal transfer 3 ml obtained on the ampicillin LB plate was picked. The expression of GST188 protein (E6/P1-P19 and E7/P1-P12) fusion protein expressed by pXXGST-2 was determined by 15% SDS-PAGE analysis. The electrophoretic mobility of the control proteins differed by about 2 kDa), and each recombinant clone was picked and sent for DNA sequencing;

3. The E6 clone with the correct sequencing result of the insert was inoculated into 3 ml of LB culture medium of Amp, and cultured overnight at 30 ° C with shaking. The next day, the fresh LB-containing LB medium was transferred at a ratio of 1/50, and cultured at 30 ° C with shaking. After 2-3 h to the concentration of 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 expression of the E7 clone was cultured at 37 ° C overnight. On the morning of the next day, the fresh Amp-containing LB medium was transferred at a ratio of 1/50, shaken at 37 °C, and induced by IPTG for 4 h.

4. 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, marked with a needle punch at the strip of the short peptide fusion protein of interest, and washed with Ponceau red with water;

5. 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 μl of anti-rabbit anti-recombinant E6 serum or positive rabbit serum was added to 8-10 ml of the reaction solution, respectively. After reacting at room temperature for 2 h, washing with PBS buffer, add 5 μl of secondary anti-rabbit anti-rabbit IgG/HRP (1:10000 dilution), react at room temperature for 1 h, wash with PBS buffer and develop color with ECL chemiluminescence kit (Figure 1 and Figure 2). ).

6. According to the results of imprinting with anti-E6 and anti-E7 antiserum (Fig. 1 and Fig. 2), it was determined that there were nine HPV18 E6 and E7 proteins respectively (E6/P1-P2, P9-P11, P15-P17 and P19). And seven (E7/P1-P6 and P12) reactive 16-polypeptides.

Example 2: Identification of two representative epitopes of HPV18E6 protein in the second round

Materials and Method:

See the corresponding part of Example 1.

The specific steps for the identification of the minimum motifs of the E6-2 and E6-5 epitopes are as follows:

1. According to the first round of E6 protein 16 polyantigenic peptide mapping results in Example 1, P1 and P19 reactive peptides were used as examples to identify their antibody recognition minimal motifs. Designing a series of 8 peptides spanning 7 amino acid residues spanning the full length sequence of P1 and P19 to encode positive and negative strands of DNA (positive strand 5'-end plus 5'-gatcc, 3'-end plus taag-3'; negative strand A DNA fragment was synthesized by adding 5'-tcgactta at the 5'-end and g-3' at the 3'-end.

The 2-4 operation steps are the same as the 2-4 steps in Example 1.

5. 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 30 μl of anti-rabbit anti-human recombinant E6 serum (1:3000) was added to 8-10 ml of the reaction solution. Dilute), react at room temperature for 2 h, wash with PBS buffer, add 5 μl 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 4A).

6. According to the three consecutive (Lanes 2-4, E6/P30-P32 in Figure 4A) and four (Lanes 3-6, E6/P91-P94 in Figure 7A) imprinted positive fragments share the residue sequence (Figure 4B and Figure 7B), the minimum motifs for the two epitopes are determined to be RELRHY and RETQV, respectively.

Based on the above method, the epitope-determined minimal motif peptide of the HPV18E6 protein was identified as shown in the following table.

Table 2 Epitope minimum motif peptide of HPV18E6 protein

SEQ ID NO.:	Amino acid sequence
		1	DPTRR
2	RELRHY
		3	NPAEKLRHL
4	HYRGQ
		5	RETQV

The antigenic peptide derived from HPV18E6 protein identified with good antibody binding ability during the experiment is shown in the following table.

Table 3 Antigen peptides of HPV18E6 protein

SEQ ID NO.:	Amino acid sequence
		14	RFEDPTRR
15	FEDPTRR
		16	EDPTRR
17	DPTRRPYK
		18	RY RELRHY
19	I RELRHY
		20	RELRHY SD
twenty one	HNPAEKLRHL
		twenty two	NPAEKLRHLN
twenty three	IAGHYRGQ
		twenty four	AGHYRGQ
25	GHYRGQ
		26	HYRGQCHS
27	QRRRETQV
		28	RRRETQV
29	RRETQV

Example 3: Identification of two representative epitopes of HPV18E7 protein in the second round

Materials and Method:

See the corresponding part of Example 1.

The specific steps for the identification of the minimum motif of E7-2 and E7-4 epitopes are as follows:

1. According to the first round of E7 protein 16 polyantigenic peptide mapping results in Example 1, P3 and P5 reactive peptides were used as examples to identify their minimal recognition of antibody recognition. Designing a series of 8 peptides spanning 7 amino acid residues spanning the full length sequence of P3 and P5 to encode positive and negative strands of DNA (positive strand 5'-end plus 5'-gatcc, 3'-end plus taag-3'; negative strand A DNA fragment was synthesized by adding 5'-tcgactta at the 5'-end and g-3' at the 3'-end.

The 2-4 operation steps are the same as the 2-4 steps in Example 1.

5. 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 30 μl of anti-rabbit anti-human recombinant E6 serum (1:3000) was added to 8-10 ml of the reaction solution. Dilute), react at room temperature for 2 h, wash with PBS buffer and add 5 μl of secondary antibody to goat anti-human IgG/HRP (1:2000 dilution), reacted at room temperature for 1 h, washed with PBS buffer and subjected to ECL chemiluminescence (Fig. 9A and Fig. 11A).

6. According to the two consecutive (Lanes 3-4, E7/P31-P32 in Figure 5A) and four (Lanes 3-6, E6/P47-P50 in Figure 6A) imprinted positive fragments share the residue sequence (Figure 9B and FIG. 11B), the minimum motifs of the two epitopes are determined to be EIPVDLL and NDEID, respectively.

Based on the above method, the epitope-determining minimal motif peptide of the HPV18E7 protein identified is shown in the following table.

Table 3 Epitope minimum motif peptide of HPV18E7 protein

SEQ ID NO.:	Amino acid sequence
		6	DIVL
7	EIPVDLL
		8	QLSDSE
9	NDEID
		10	HQHL
11	CASQQ

The antigenic peptide derived from HPV18E7 protein identified with good antibody binding ability during the experiment is shown in the following table.

Table 5 antigenic peptides of HPV18E7 protein

SEQ ID NO.:	Amino acid sequence
		30	ATLQDIVL
31	TLQDIVLH
		32	LQDIVLHL
33	QDIVLHLE
		34	DIVLHLEP
35	N EIPVDLL
		36	EIPVDLL C
37	HEQLSDSE
		38	EQLSDSE
39	QLSDSEEE
		40	EEE NDEID
41	EE NDEID G
		42	E NDEID GV
43	NDEID GVN
		44	DGVNHQHL
45	GVNHQHLP
		46	VNHQHLPA
47	NHQHLPAR
		48	HQHLPARR
49	CPWCASQQ
		50	PWCASQQ
51	WCASQQ

Although the present invention describes a total of 11 epitope minimal motif peptides on the HPV18 oncogenic E6 and E7 proteins, and the development of therapeutic properties alone or in combination with polypeptides containing minimal motifs (eg, 8 peptides) or polypeptide fusion proteins The HPV multi-epitope vaccine may be used alone or in combination as an antigen for clinical detection of human HPV type 18 infection (these antigens may be chemically synthesized or fused), but after reading the above teachings of the present invention, those skilled in the art Various obvious variations can be made to the epitope peptide motifs of the invention and the extended minimal matrix-containing polypeptide fusion proteins without departing from the spirit and scope of the invention, and such equivalent forms are also within the scope of the present application. The scope defined by the claims is defined. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the

Claims (10)

1. An epitope minimal motif peptide of human papillomavirus type 18 E6 protein, characterized in that said minimal epitope motif peptide has the amino acid sequence shown in SEQ. ID No. 1, and is designated HPV18 E6-1 ^{6- 10;} or having an amino acid sequence shown in SEQ.ID No.2, referred to as HPV18 E6-2 ^76-81; or an amino acid sequence shown in SEQ.ID No.3, referred to as HPV58 E6-3 ^113-121; or a The amino acid sequence shown in SEQ. ID No. 4, which is designated as HPV58 E6-4 ^133-137 ; or has the amino acid sequence shown in SEQ. ID No. 5, is designated HPV58 E6-5 ^154-158 .
2. An epitope minimal motif peptide of human papillomavirus type 18 E7 protein, characterized in that said minimal epitope motif peptide has the amino acid sequence shown in SEQ. ID No. 6, and is designated HPV18 E7-1 ^{10- 13} ; or have the amino acid sequence shown in SEQ. ID No. 7, which is designated as HPV18 E7-2 ^20-26 ; or has the amino acid sequence shown in SEQ. ID No. 8, which is designated as HPV18 E7-3 ^30-35 ; or The amino acid sequence shown in SEQ. ID No. 9 is designated as HPV18 E7-4 ^38-42 ; or has the amino acid sequence shown in SEQ. ID No. 10, which is designated as HPV18 E7-5 ^46-49 ; or has SEQ. ID No. The amino acid sequence shown in .11 is referred to as HPV18E7-6 ^101-105 .
3. A polypeptide comprising the epitope minimal motif peptide of claim 1 wherein the polypeptide:

   An amino acid sequence represented by X ₁ X ₂ DPTRRX ₃ , designated HPV18 E6-1-1 (preferably as SEQ. ID No. 12), wherein X ₁ , X ₂ , and X ₃ are independently Non-specific amino acid residues are either none; or

   Having the amino acid sequence of X ₁ RELRHYX ₂ , designated HPV18 E6-2-1 (preferably as shown in SEQ. ID No. 13), wherein X ₁ and X ₂ are independently non-specific amino acid residues, respectively. Or no; or

   An amino acid sequence represented by X ₁ NPAEKLRHLX ₂ , designated HPV18 E6-3-1 (preferably as SEQ. ID No. 14), wherein X ₁ and X ₂ are independently non-specific amino acid residues, respectively. Or no; or

   An amino acid sequence represented by X ₁ X ₂ HYRGQX ₃ , designated HPV18 E6-4-1 (preferably as SEQ. ID No. 15), wherein X ₁ , X ₂ , and X ₃ are independently Non-specific amino acid residues are either none; or

   An amino acid sequence represented by X ₁ X ₂ X ₃ RETQV, designated HPV18 E6-5-1 (preferably as SEQ. ID No. 16), wherein X ₁ , X ₂ , and X ₃ are independently It is a non-specific amino acid residue or is none.
4. A polypeptide comprising the epitope minimal motif peptide of claim 2, wherein the polypeptide has:

   Having the amino acid sequence of X ₁ X ₂ DIVLX ₃ X ₄ , designated HPV18 E7-1-1 (preferably as shown in SEQ. ID No. 17), in the sequence of X ₁ , X ₂ , X ₃ , and X ₄ are independently non-specific amino acid residues or none; or

   An amino acid sequence represented by X ₁ EIPVDLLX ₂ , designated HPV18 E7-2-1 (preferably as SEQ. ID No. 18), wherein X ₁ and X ₂ are independently non-specific amino acid residues, respectively. Or no; or

   An amino acid sequence represented by X ₁ QLSDSEX ₂ , designated HPV18 E7-3-1 (preferably as SEQ. ID No. 19), wherein X ₁ and X ₂ are independently non-specific amino acid residues, respectively. Or no; or

   An amino acid sequence represented by X ₁ X ₂ NDEIDX ₃ , designated HPV18 E7-4-1 (preferably as SEQ. ID No. 20), wherein X ₁ , X ₂ , and X ₃ are independently Non-specific amino acid residues are either none; or

   An amino acid sequence represented by X ₁ X ₂ HQHLX ₃ X ₄ , designated HPV18 E7-5-1 (preferably as SEQ. ID No. 21), in the sequence of X ₁ , X ₂ , X ₃ , and X ₄ are independently non-specific amino acid residues or none; or

   An amino acid sequence represented by X ₁ X ₂ X ₃ CASQQ, designated HPV18 E7-6-1 (preferably as SEQ. ID No. 22), wherein X ₁ , X ₂ , and X ₃ are independently It is a non-specific amino acid residue or is none.
5. An epitope-less minimal motif peptide of human papillomavirus (HPV) type 18, said motif peptide being derived from E6 or E7 protein of papillomavirus type 18, and said motif peptide having a length of ≤ 9 An amino acid, and the motif peptide has an activity of binding to an anti-HPV18 type antibody; preferably, the motif peptide is selected from the group consisting of:

   DPTRR (SEQ ID NO.: 1), RELRHY (SEQ ID NO.: 2), NPAEKLRHL (SEQ ID NO.: 3), HYRGQ (SEQ ID NO.: 4), RETQV (SEQ ID NO.: 5), DIVL (SEQ ID NO.: 6), EIPVDLL (SEQ ID NO.: 7), QLSDSE (SEQ ID NO.: 8), NDEID (SEQ ID NO.: 9), HQHL (SEQ ID NO.: 10) and CASQQ (SEQ ID NO.: 11).
6. An antigenic peptide of human papillomavirus (HPV) type 18, characterized in that the antigenic peptide structure is as shown in formula I:

   Ya-Y-Yb, I,

   And meet the following characteristics:

   (a) In the formula I, Y is the motif peptide of claim 5;

   (b) In the formula I, Ya and Yb are independently a fragment consisting of no 1, 2, or 3 amino acids, and the total number of Y1 and Y2 amino acids is ≤4;

   (c) the antigen peptide has an activity of binding to an anti-HPV antibody;

   Wherein "-" means a peptide bond or a peptide linker; preferably, the antigenic peptide is selected from the group consisting of:

   Y ₁ Y ₂ DPTRRY ₃ , Y ₁ RELRHYY ₂ , Y ₁ NPAEKLRHLY ₂ , Y ₁ Y ₂ HYRGQY ₃ , Y ₁ Y ₂ Y ₃ RETQV,

   Y ₁ Y ₂ DIVLY ₃ Y ₄ , Y ₁ EIPVDLLY ₂ , Y ₁ QLSDSEY ₂ , Y ₁ Y ₂ NDEIDY ₃ , Y ₁ Y ₂ HQHLY ₃ Y ₄ , and Y ₁ Y ₂ Y ₃ CASQQ, wherein each Y ₁ , Y ₂ , Y ₃ , and Y ₄ represent an amino acid residue, respectively.
7. A fusion protein comprising the motif peptide of claim 5 or the antigenic peptide of claim 6, and a vector sequence derived from a non-HPV protein, and an optional tag sequence.
8. A composition characterized in that it contains:

   (a) one or more peptide fragments selected from the group consisting of:

   The motif peptide according to claim 1, 2 or 5, the polypeptide according to claim 3, the antigenic peptide of claim 6, and the fusion protein of claim 7;

   (b) a pharmaceutically acceptable carrier or excipient.
9. An isolated collection of polypeptides, characterized in that said collection of polypeptides consists of two or more polypeptides selected from the group consisting of one or more of the following:

   (i) one or more motifs selected from the group consisting of claim 5; and/or

   (ii) one or more antigenic peptides selected from claim 6; and/or

   (iii) one or more fusion proteins selected from the group consisting of claim 7.
10. The motif peptide according to claim 1, 2 or 5, the polypeptide according to claim 3, the antigen peptide according to claim 6, the fusion protein according to claim 7, or the composition according to claim 8. Use of the polypeptide set of claim 9, or a reagent or kit for detecting an HPV18 type E6 and/or E7 early protein-inducing antibody; or

    For the preparation of a medicament for the prevention or treatment of HPV type 18 infection; or

    For the preparation of a medicament for preventing or treating a disease caused by an HPV type 18 infection; or

    It is used to detect E6 and/or E7 protein antibodies against HPV18.

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