WO2014127741A1 - Antigen for detecting anti-human papilloma virus antibodies, test kit and applications thereof - Google Patents

Abstract

Provided is an antigen peptide used for detecting anti-human papilloma virus (HPV) antibodies, said antigen peptide being derived from the HPV E7 protein. Said proteins can be used to test for anti-HPV antibodies in blood or in saliva. Also provided, therefore, are a composition and test kit comprising said antigenic peptides.

Description

 Antigen, kit and application thereof for detecting anti-human papillomavirus antibody

 The invention relates to the technical field of antibody detection, in particular to the technical field of detection of anti-human papillomavirus (HPV) antibody, in particular to an antigen for detecting anti-human papillomavirus antibody, related immunoassay kit and the same Applications, especially for the high-risk human papillomavirus (HPV) subtypes HPV16 and HPV18. Background technique

 Cervical cancer is the second most common female malignancy, and human papillomavirus (HPV) is a necessary condition for its occurrence. Although HPV infection is common in the population, such as: Women with high incidence of cervical cancer still have an infection rate of 20.8% after the age of 35. However, more than 80% of HPV DNA-positive women do not progress to cancer or precancerous lesions. Therefore, HPV DNA detection defects are incapable of distinguishing between transient infections and infections that can cause cervical lesions, resulting in higher false positive rates. Numerous studies have shown that HPV oncogene expression products (oncoprotein oncoprotein) have high carcinogenic activity, so these biomarkers can be used as predictors of cervical cancer that are more accurate and specific than HPV DNA. The expression levels of high-risk HPV E2, E6, and E7 oncoproteins are directly related to different stages of cervical precancerous lesions. Using this feature, a new molecular screening index for cervical cancer can be constructed.

 Studies on the carcinogenic mechanism of HPV have shown that the early expression proteins E6 and E7 of high-risk HPV play an important role in the development of cervical cancer. HPV E6 and E7 proteins are generally overexpressed in cervical epithelial cells of cervical lesions and cancer patients, and persistently expressed and overexpressed E6, E7 oncoproteins interact with host cell regulatory proteins (eg, P53 and Rb proteins) Inactivates tumor suppressor, leading to immortalization and malignant transformation of cells.

 In addition to HPV DNA testing and HPV oncoprotein detection, HPV antibodies in blood (body fluids) have also been shown to be highly relevant for HPV infection and the history of cervical cancer. HPV L1 blood antibodies can be detected by standard ELISA methods to provide HPV infection information. As HPV infection time prolongs, HPV induces anti-HPV antibodies, so anti-HPV antibodies in serum can be detected. Wikstrom et al tested serum IgG antibodies to HPV6 and HPV11 capsid proteins in 47 male patients with condyloma acuminata and 32 men with a history of condyloma acuminata, and compared with 205 men with no history of genital condyloma acuminata. As a result, the anti-HPV6 type IgG antibody in the serum was 35% in men who had a history of condyloma acuminata, 27% in the current condyloma acuminata, and 10% in the control group. This indicates that the occurrence of anti-HPV6 IgG antibodies occurred later in the condyloma acuminata, and IgG positive also reflected that the virus had been infected.

 Stone et al reported the use of this method for the infection rate of 7218 HPV-16 in the general population of the United States. The results showed that the female positive rate was 17.9% and the male positive rate was 7.9%. The HPV L1 antibody ELISA method has been widely used in the development and clinical trials of the HPV vaccine (Merck Gardasil preventive vaccine). Luevano et al. used a high-throughput assay for serum samples from patients with cervical cancer, and analyzed antibody profiling of a total of 54604 proteins in 13 HPV types. The results showed that antibodies to carcinogenic proteins in the blood and cervical cancer, high lesions, Low-grade lesions, and asymptomatic people have a strong correlation (Luevano, M., et al, High-Throughput Profiling of the Humoral Immune Responses Against Thirteen Human Papillomavirus Types by Proteome Microarrays. 2010. Virology. 2010; 405 ( 1): 31-40).

Therefore, in order to achieve high sensitivity, high specificity, and high stability of HPV antibody detection, it is required to provide a novel antigen for detecting antibodies against human papillomavirus. Summary of the invention

 In order to solve the above problems, an object of the present invention is to provide an antigen peptide for detecting an anti-human papillomavirus antibody and a related immunoassay kit, which can detect antigen peptides against human papillomavirus antibodies with high sensitivity Highly specific and highly stable detection of HPV antibodies in blood, saliva or other samples, which can provide a basis for doctors to accurately diagnose cervical intraepithelial neoplasia and cervical cancer, and then timely take treatment to prevent cancer , or the spread of cancer, timely relief of patient suffering, the immunoassay kit made of it can quickly and accurately detect HPV antibodies in blood, saliva or other samples, suitable for large-scale application. In a first aspect of the invention, a human papillomavirus antigen peptide is provided, the antigen peptide is derived from an E7 protein of human papillomavirus, and the antigen peptide is 5-50 amino acids in length, and The antigenic peptide can bind to an antibody against human papillomavirus. The antigenic peptide may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 amino acids in length.

 In another preferred embodiment, the antibody comprises a monoclonal antibody, a polyclonal antibody or an antiserum. In another preferred embodiment, the m+2th position, the m+3th position, the m+5th position, and the m+6th position of the antigenic peptide are Asp, Ser, Glu, Glu, and/or m+8. The -m+12 amino acids are in turn Asp, Glu, Ile, Asp and Gly, where m ≥ 0 and m is an integer. In another preferred embodiment, the structure of the antigenic peptide is as shown in Formula I

 [XaaO]-[Xaal]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6] (I)

 In the formula,

 XaaO is a peptide consisting of none or 1-5 amino acids;

 Xaal is DS;

 Xaa2 is selected from S or E;

 Xaa3 is EE;

 Xaa4 is selected from E or N;

 Xaa5 is DEIDG;

 Xaa6 is a peptide consisting of no or a length of 1 to 30 amino acids.

 In another preferred embodiment, XaaO and/or Xaa6 are not none.

 In another preferred embodiment, the peptide sequence of XaaO and/or Xaa6 is an E7 protein sequence derived from HPV.

 In another preferred embodiment, for XaaO and/or Xaa6, the "HPV-derived E7 protein sequence" means that the peptide sequence of XaaO is derived from the sequence of the E7 protein of HPV and directly upstream of Xaal and The peptide sequence of Xaa6 is derived from the sequence of the E7 protein of HPV and directly downstream of Xaa5.

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

 In another preferred embodiment, the HPV virus comprises HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68.

 In another preferred embodiment, XaaO is a peptide of 1, 2 or 3 amino acids, such as &&0 being 8, LN or IDG. In another preferred embodiment, Xaa6 is a peptide consisting of more than 10 (e.g., 10-20, preferably 11-18) amino acids in length. In another preferred embodiment, said Xaa6 is at positions 14-24 of SEQ ID NO.: 3.

 In another preferred embodiment, the Xaa6 is at positions 13-28 of SEQ ID NO.: 5.

In another preferred embodiment, the antigenic peptides include both salt and non-salt forms. In another preferred embodiment, the salt is pharmaceutically acceptable In another preferred embodiment, the structure of the antigenic peptide is as shown in Formula II,

 [YaaO]-[Yaal]-[Yaa2]-[Yaa3]-[Yaa4]-[Yaa5] (II)

 In the formula,

 YaaO is a peptide consisting of no, or 1-30 amino acids;

 Yaal is DS;

 Yaa2 is selected from S or E;

 Yaa3 is EE;

 Yaa4 is selected from E or N;

 Yaa5 is a peptide consisting of no, or 1-30 amino acids in length.

 In another preferred embodiment, YaaO and/or Yaa5 are not none.

 In another preferred embodiment, the YaaO is a peptide consisting of 5-25 amino acids and Yaa5 is a peptide consisting of 1-10 amino acids. Preferably, YaaO is a peptide consisting of 10-25 amino acids and Yaa5 is a peptide consisting of 1-10 amino acids.

 In another preferred embodiment, the YaaO is a peptide consisting of 1-5 amino acids and Yaa5 is a peptide consisting of 10-25 amino acids. Preferably, YaaO is a peptide consisting of 1-5 amino acids and Yaa5 is a peptide consisting of 15-25 amino acids.

 In another preferred embodiment, the peptide sequence of YaaO and/or Yaa5 is an E7 protein sequence derived from HPV.

 In another preferred embodiment, the "E7 protein sequence derived from HPV" refers to the sequence of the YaaO peptide sequence derived from the E7 protein of HPV and directly upstream of Yaa 1 for YaaO and/or Yaa5. The peptide sequence of and/or Yaa5 is derived from the sequence of the E7 protein of HPV and located directly downstream of Yaa4.

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

 In another preferred embodiment, the HPV virus comprises HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 68.

 In another preferred embodiment, the antigenic peptides include both salt and non-salt forms. In another preferred embodiment, the salt is a pharmaceutically acceptable salt. In another preferred embodiment, the antigenic peptide is selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 5, or a combination thereof. In another preferred embodiment, the antigenic peptide is selected from the group consisting of:

 (a) a polypeptide having the amino acid sequence shown in any one of SEQ ID ΝΟ: 1-5;

 (b) a polypeptide having > 80% homology to the amino acid sequence shown in any one of SEQ ID ΝΟ: 1-5, and having the ability to bind to an antibody against human papillomavirus;

 (c) Derivation of the amino acid sequence of any one of SEQ ID ΝΟ: 1-5 by substitution, deletion or addition of 1-5 amino acid residues, and retaining the ability to bind to antibodies against human papillomavirus Peptide.

 In another preferred embodiment, the antibody against human papillomavirus is an antibody against the HPV virus E7 protein.

In another preferred embodiment, the antigenic peptide is obtained by chemical synthesis or bioengineering methods, including recombinant proteins, fusion proteins. In a second aspect of the invention, there is provided a fusion protein comprising the antigenic peptide of the first aspect of the invention and an optional tag sequence. In a third aspect of the invention, there is provided an isolated nucleic acid molecule encoding an antigenic peptide according to the first aspect of the invention or a fusion protein according to the second aspect of the invention. According to a fourth aspect of the invention, there is provided a composition, characterized in that it comprises:

 (a) the human papillomavirus antigen peptide of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a fusion protein according to the second aspect of the invention;

 (b) a pharmaceutically acceptable carrier or excipient.

 In another preferred embodiment, the composition includes a pharmaceutical composition and a vaccine composition. According to a fifth aspect of the present invention, there is provided the use of the human papillomavirus antigen peptide according to the first aspect of the present invention, which is characterized in that it is used for preparing a reagent or a kit for detecting HPV; or

 For the preparation of a medicament for the prevention or treatment of human papillomavirus infection; or

 For detection of anti-human papillomavirus antibodies.

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

 In another preferred embodiment, the drug is selected from a vaccine or an antibody.

 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 sixth aspect of the invention, there is provided an immunoassay kit comprising the antigen peptide for detecting an anti-human papillomavirus antibody according to the first aspect of the invention . The kit may include one or more antigenic peptides for detecting anti-human papillomavirus antibodies according to the first aspect of the invention.

 In another preferred embodiment, the kit further comprises a vector, and the antigenic peptide for detecting an anti-human papillomavirus antibody is coated on the carrier.

 In another preferred embodiment, the immunodetection kit further comprises a labeled secondary antibody against human papillomavirus antibody. The secondary antibody is used to detect antibodies in human serum IgG, IgM, or IgA. The label used may be horseradish peroxidase, alkaline phosphatase, fluorescent molecule FITC (or other fluorescent label), chemiluminescence detection. The detection method may be qualitative, quantitative analysis by chromogenic method, fluorescence method, chemiluminescence, and electrochemiluminescence.

In another preferred embodiment, the secondary antibody to the labeled anti-human papillomavirus antibody is a secondary antibody to a horseradish peroxidase-labeled anti-human papillomavirus antibody. It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described in the following (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. The invention has the beneficial effects that the patient can detect the specific antibody IgM, IgA or IgG molecules in the serum and oral saliva with high specificity and sensitivity after being infected by the virus, so that the doctor can accurately diagnose the cervical epithelium of the patient. The tumor detection and cervical cancer bring the basis, and then timely treatment to prevent cancer, or the spread of cancer, and timely relief of the patient's pain, the immunoassay kit can be quickly and accurately detected in blood or saliva. HPV resistance Body, suitable for large-scale promotion and application. detailed description

 The inventors have extensively and intensively studied, designed and synthesized dozens of human papillomavirus antigen peptides having different sequences, and selected antigen peptides having excellent sensitivity and specificity. Specifically, the inventors applied bioinformatics methods, based on homology analysis and biological characteristics (including immunogenicity), and designed dozens of candidate sequences, which were synthesized and separated by solid phase method. Purification yields high purity polypeptide fragments. For these candidate sequences, peptides were used as ELISA plate antigens, and patient serum (up to 546 patients with cervical cancer and cervical lesions) and healthy subjects (600 cases) were used for detection; and HPV molecular test results and TCT cell morphology were compared. Academic results, pathological diagnosis results, statistical analysis of serum test data. After repeated screening, a new class of antigenic peptides that bind to anti-human papillomavirus HPV oncoprotein antibodies were obtained. It can be used to detect, prevent and treat human papillomavirus infections, and related cancers. The present invention has been completed on this basis. Active polypeptide

 As used herein, the terms "HPV oncoprotein antigen", "HPV oncoprotein antigen peptide", "human papillomavirus oncoprotein antigen peptide", "human papillomavirus oncoprotein antigen", "antigen of human papillomavirus antibody" "Peptide antigen" is used interchangeably. The polypeptide sequence is derived from a polypeptide of the human papillomavirus (e.g., HPV16, HPV18) E7 protein.

 As used herein, the terms "HPV antigen of the present invention", "HPV antigen peptide of the present invention", "human papillomavirus antigen peptide of the present invention", "human papillomavirus antigen peptide of the present invention" are used interchangeably and mean An antigen peptide that binds to an anti-human papillomavirus antibody (such as the polypeptides shown in SEQ ID ΝΟ: 1 to 5). These antigenic peptides can be used to detect oncoprotein antibodies induced by HPV oncogene products. HPV oncoprotein is a cancer cell protein marker that is expressed when HPV virus invades cells. Further, the term also includes variant forms of the sequence of SEQ ID NO: 1-5 having the function of an antibody that binds to anti-human papillomavirus. These variants include, but are not limited to, 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions at the C-terminus and/or N-terminus or One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is usually not altered. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the structure and function of the protein. Furthermore, the term also encompasses polypeptides of the invention in both monomeric and multimeric forms. The term also includes both linear as well as non-linear polypeptides (e.g., cyclic peptides).

 The invention also encompasses active fragments, derivatives and analogs of human papillomavirus antigenic peptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of binding to an antibody against human papillomavirus. A polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one of the amino acid residues, or (iii) a polypeptide formed by fusion of the antigen peptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide formed by fusion of the polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

A preferred class of reactive derivatives means that up to 3, preferably up to 2, and more preferably up to 1 amino acid are replaced by amino acids of similar or similar nature to the amino acid sequence of Formula I or Formula II. Peptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1. Table 1

The invention also provides analogs of human papillomavirus antigen peptides. The difference between these analogs and the polypeptides shown in SEQ ID NOS: 1 to 5 may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof. Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, Υ-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

 Modifications (usually without altering the primary structure) include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.

The polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. These salts include, but are not limited to, salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, hydrazine sulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, amino phthalates or other conventional "prodrugs". Coding sequence

 The invention also relates to polynucleotides encoding human papillomavirus antigen peptides.

 The polynucleotide of the present invention may be in the form of DNA or the form of R A . The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the sequence encoding the polypeptides shown in SEQ ID NOS: 1 to 5 or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a polypeptide having the sequence of SEQ ID ΝΟ: 1 to 5, but differs in the corresponding coding region sequence.

 The full-length nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. At present, it has been possible to obtain a DNA sequence encoding the polypeptide of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

 The invention also relates to vectors comprising the polynucleotides of the invention, and host cells produced by genetic engineering using the vectors or polypeptide coding sequences of the invention. Preparation

 The polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.

 A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods. Solid phase synthesis allows rapid sample acquisition, and the appropriate resin carrier and synthesis system can be selected based on the sequence characteristics of the peptide of interest. For example, a preferred solid phase support in the Fmoc system is a Wang resin with a C-terminal amino acid attached to the peptide, a Wang resin structure is polystyrene, and an arm between the amino acids is 4-alkoxybenzyl alcohol; using 25% hexahydropyridine /Dimercaptoamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus according to the given amino acid sequence. After the completion of the synthesis, the synthesized proinsulin-related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-nonylphenol, and the protecting group was removed. The resin was removed by filtration and the diethyl ether was precipitated to obtain a crude peptide. After the solution of the obtained product was lyophilized, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When the Boc system is used for solid phase synthesis, it is preferred that the resin be a PAM resin to which a C-terminal amino acid is linked, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxydecyl phenylacetamide; In the system, in a deprotection, neutralization, coupling cycle, the protecting group Boc was removed with TFA/dichlorodecane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane). After the condensation is completed, the peptide chain is cleaved from the resin by treatment with hydrogen fluoride (HF) containing p-nonylphenol (5-10%) at 0 ° C for 1 hour while removing the protecting group. 50-80% The peptide is extracted with acetic acid (containing a small amount of thiol ethanol), and the solution is further lyophilized and further purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. It can be used in the field of peptide chemistry. Various coupling agents and coupling methods are coupled to each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea can be used. Direct coupling of hexafluorophosphate (HBTU). Purity and structure of synthetic short peptides Confirmed by RP-HPLC analysis and mass spectrometry.

 In a preferred embodiment, the human papillomavirus antigen peptide of the present invention is prepared by solid phase synthesis according to its sequence, and purified by high performance liquid chromatography to obtain a high-purity peptide freeze-dried powder, which is stored at -20 °C.

 Another method is to produce a polypeptide of the invention using recombinant techniques. The polynucleotide of the present invention can be utilized to express or produce the human papillomavirus antigen peptide of the present invention by conventional recombinant DNA techniques. Generally there are the following steps:

(1) using a polynucleotide (or variant) encoding a human papillomavirus antigen peptide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide; (2) a host cell cultured in a suitable medium;

 (3) Separating and purifying proteins from the culture medium or cells.

 The recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 Since the polypeptide of the present invention is short, it is conceivable to connect a plurality of polypeptides in series, to obtain an expression product in a multimeric form after recombinant expression, and then to form a desired small peptide by enzymatic cleavage or the like. Pharmaceutical composition and method of administration

 The antigenic peptide or fusion protein of the present invention can induce an antibody against HPV or HPV-positive cells (cancer cells) and lymphocyte T cell activity as an immunogen in vivo, thereby achieving an anticancer therapeutic effect. Further, the antigen peptide of the present invention has excellent specificity and immunological activity, and thus can be used for preparing an anti-cancer vaccine for immunotherapy or prevention.

 In another aspect, the present invention provides a pharmaceutical composition (including a vaccine composition) comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof (or a coding sequence thereof); b) a pharmaceutically acceptable carrier or excipient. The amount of the polypeptide of the present invention is usually from 10 μg to 100 mg / dose, preferably from 100 to 1000 μg / dose.

 For the purposes of the present invention, an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg of body weight of the polypeptide of the invention. Furthermore, the polypeptide of the present invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

 The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

 The pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

 In general, the therapeutic composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be prepared as a solid form of liquid carrier suitable for administration in solution or suspension prior to injection.

 Once formulated into a composition of the invention, it can be administered by conventional routes including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal or topical administration. The subject to be prevented or treated may be an animal; especially a human.

 When the pharmaceutical composition of the present invention is used for actual treatment, a pharmaceutical composition of various dosage forms can be used depending on the use. It is preferably an injection.

 These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in the usual manner according to the dosage form.

The pharmaceutical compositions of the invention may also be administered in the form of sustained release agents. For example, a human papillomavirus antigen peptide or a salt thereof can be incorporated The pellet or microcapsule is loaded with a sustained-release polymer, and the pellet or microcapsule is then surgically implanted into a human tissue. Examples of the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, mercaptocellulose, lactic acid polymer, A lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.

 When the pharmaceutical composition of the present invention is used for prevention or treatment, the dose of the human papillomavirus antigen peptide or a pharmaceutically acceptable salt thereof as an active ingredient may be according to each subject (patient) to be prevented or treated. The weight, age, sex, and degree of symptoms are reasonably determined. The main advantages of the invention include:

 (a) The human papillomavirus antigen peptide of the present invention has the ability to specifically bind to an anti-human papillomavirus antibody, and has high sensitivity and specificity;

 (b) The human papillomavirus antigen peptide of the present invention is moderate in length and easy to prepare;

 (c) Easy detection of blood, saliva or other body fluids. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and 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 usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Example 1

I. Materials and methods

1, reagents and drugs

 Horseradish peroxidase labeled Goat Anti-Human IgG (FC), purchased from Priece Product # 31413. 2, instruments and supplies

 MULTISKAN MK3 microplate reader (Thermo); ELISA plate (COSTAR).

3, the main solution system

 • Coating buffer (PBS phosphate solution): Potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 12g 2.9g, sodium chloride 8.0g, potassium chloride 0.2g, distilled water to 1L, 4 degrees storage.

 • Wash buffer (PBS-Tween 20, 0.1%): Tween-20 lmL, add to 1L of the dilution buffer (PBS), mix and mix, adjust the pH to 7.0 ~ 7.2.

 • Blocking solution: 5%milk-PBS-Tween

 • Serum dilution: 10% FBS-PBS-Tween

 TMB display liquid: Amresc, CAT NO.64285730, before use TMB background color development A liquid and B liquid volume mixing.

 o Substrate coloring Liquid A: Sodium acetate 13.6g, citric acid 1.6g, hydrogen peroxide (30%) 0.3ml, distilled water added to 500ml.

o Substrate coloring liquid B: disodium edetate 0.2g citric acid 0.95g glycerol 50ml tetraterpene Aniline 0.2 g of distilled water was added to 500 ml.

 • Stop solution (2mol/L H2S04): Double distilled water 178.3mL, add concentrated 4 acid (98%) 21.7 1^.

4. Establishment of an indirect ELISA method

 1) Dilute HPV peptide antigen with PBS HPV16-1/HPV16-2/HPV16-3/HPV18-1/HPV18-2 concentration is

4ug/ml, coated costar slats lOOul/well, 4°C overnight.

 2) Wash once with PBS and pat dry.

 3) Add 5% milk powder - PBS closed, 250 ul / well, 37 degrees 3 hours.

 4) Wash once with PBST and pat dry.

 5) 10% FBS-PBST diluted serum 1 : 50 dilution, shake the shaker.

 6) Add diluted serum, blank control, negative control and positive control, 50 ul / well, incubate for 1 hour at 37 degrees.

7) Wash 5 times with 0.1% tween-PBS and pat dry.

 8) Add horseradish peroxidase-labeled goat anti-human, dilute with 10% FBS-PBST (1: 40000), lOOul/well, and incubate for 37 minutes at 37 degrees.

 9) Wash 5 times with 0.1% tween-PBS and pat dry.

 10) TMB color development: TMB A liquid and B liquid are mixed in equal volume, lOOul/well, 37 degrees 20 min.

11) 2M H ₂ S0 ₄ , 50 ul/well terminated, read at 450 nm with a microplate reader.

5. Kit composition:

 This kit consists of the following parts:

 1. A costar ELISA plate coated with an antigen for detecting an anti-human papillomavirus antibody of the present invention

 2. Horseradish peroxidase labeled sheep anti-human tube (lOul)

 3. 10*PBST—bottle (10ml)

 4. TMB coloring solution A bottle (6ml), one bottle of sputum (6ml)

 5. One bottle of stop solution (6ml)

6, coated antigen

 The polypeptide antigen is as follows

 SEQ ID

 Name sequence note

 NO.:

 The 16E7 protein

HPV16-1 KCDSTLRLCVQSTHVDIRTLE 1

 60-80

The 16E7 protein

HP VI 6 -2 PTLHEYMLDLQPETTDLYCYEQLNDSSEEE 2

 6-35

 The 16E7 protein

HPV16-3 LNDSSEEEDEIDGPAGQAEPDRAH 3

 28-51

The 18E7 protein

HPV18-1 IDGVNHQHLPARRAEPQR 4

 41-58

HPV18-2 SDSEEENDEIDGVNHQHLPARRAEPQRH 5 18E7 protein 32-59

The sequence alignment of SEQ ID NO.: 2-5 is as follows:

 SEQ ID NO. : 2: PTLHEYMLDLQPETTDLYCYEQLNDSSEEE

 SEQ ID NO. : 3 : LNDSSEEEDEIDGPAGQAEPDRAH

 SEQ ID NO. : 4 : IDGVNHQHLPARRAEPQR

 SEQ ID NO. : 5 : SDSEEENDEIDGVNHQHLPARRAEPQRH II. Test results

 The kit selects 5 polypeptides (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) as coating selection, serum is detected by ELISA method, and each record is recorded. The OD value of each sample (450 nm wavelength).

 1. The healthy human serum was detected by ELISA using an enzyme-labeled plate coated with an antigen, and the raw data of the test were as follows.

 Table 1: HPV blood ELISA test normal human serum results table

Serum number SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 Normal human No. 1 0.237 0.232 0.300 0.367 0.237 Normal person No. 2 0.320 0.238 0.384 0.503 0.342 Normal person No. 3 0.427 0.319 0.529 0.484 0.364 Normal person No. 4 0.373 0.265 0.401 0.991 0.882 Normal person No. 5 0.338 0.198 0.367 0.606 0.468 Normal person No. 6 0.276 0.227 0.391 0.251 0.221 Normal person No. 7 0.232 0.214 0.311 0.278 0.252 Normal person No. 8 0.284 0.267 0.406 0.506 0.289 Normal Human No. 9 0.286 0.283 0.419 0.382 0.365 Normal person No. 10 0.226 0.168 0.249 0.229 0.169 Normal person No. 11 0.266 0.213 0.320 0.378 0.275 Normal person No. 12 0.303 0.212 0.389 0.422 0.312 Normal person No. 13 0.250 0.262 0.402 0.575 0.345 Normal person No. 14 0.343 0.277 0.179 0.278 0.243 Normal person No. 15 0.422 0.169 0.129 0.357 0.242 Normal person No. 16 0.372 0.132 0.127 0.307 0.306 Normal person No. 17 0.729 0.272 0.274 0.664 0.420 Normal person No. 18 0.502 0.390 0.229 0.437 0.349 Normal person No. 19 0.526 0.383 0.241 0.461 0.309 Normal person No. 20 0.482 0.508 0.265 0.417 0.339 Normal person 21 No. 0.584 0.446 0.395 0.519 0.415 Normal 22 0.539 0.451 0.319 0.474 0.410 Normal person No. 23 0.500 0.294 0.205 0.435 0.303 Normal person No. 24 0.472 0.372 0.253 0.407 0.294 Normal person No. 25 0.616 0.405 0.265 0.551 0.373 Normal person No. 26 0.704 0.480 0.430 0.639 0.626 Normal person No. 27 0.519 0.335 0.288 0.454 0.247 Normal person No. 28 0.750 0.450 0.359 0.685 0.374 Normal person No. 29 0.549 0.359 0.270 0.484 0.242 Normal person No. 30 0.333 0.261 0.275 0.277 0.217 Normal person No. 31 0.277 0.192 0.262 0.329 0.301 Normal person No. 32 0.375 0.283 0.369 0.474 0.376 Normal person No. 33 0.264 0.201 0.212 0.281 0.205 Normal Person No. 34 0.293 0.257 0.312 0.275 0.255 Normal person 35 No. 0.323 0.267 0.449 0.291 0.201 Normal person No. 36 0.280 0.256 0.354 0.310 0.281 Normal person No. 37 0.171 0.137 0.160 0.398 0.285 Normal person No. 38 0.267 0.180 0.190 0.475 0.455 Normal person No. 39 0.201 0.123 0.163 0.370 0.215 Normal person No. 40 0.202 0.187 0.276 0.473 0.273 Normal person No. 41 0.206 0.298 0.119 0.181 0.183 Normal person No. 42 0.288 0.455 0.155 0.263 0.233 Normal person No. 43 0.224 0.186 0.213 0.296 0.255 Normal person No. 44 0.219 0.206 0.243 0.184 0.398

2. Two polypeptides (SEQ ID NO: 3, SEQ ID NO: 5) were selected as the coated and blocked ELISA plate to detect normal human serum by ELISA. The raw data of the test were as follows;

 Table 2: HPV blood ELISA test normal human serum results table

 Serum number SEQ ID NO: 3 SEQ ID NO: 5 Serum number SEQ ID NO: 3 SEQ ID NO: 5

N B1 0.562 0.575 N B101 0.947 0.989

N B2 No. 0.232 0.436 N B102 No. 0.650 0.606

N B4 0.894 0.766 N B103 0.464 0.618

N B5 0.573 0.415 N B104 0.777 0.327

N B6 0.768 0.644 N B105 0.605 0.669

N B7 0.643 0.538 N B106 0.347 0.342

N B8 0.717 0.621 N B107 0.642 0.626

N B9 0.649 0.509 N B108 1.125 0.958

N B10 0.514 0.477 N B109 0.517 0.480

N B11 No. 0.653 0.558 N B110 No. 0.703 0.638 NB12 No. 0.983 1.006 NB111 No. 0.769 0.725

NB13 0.745 0.635 NB112 0.543 0.414

NB14 0.423 0.340 NB113 0.318 0.410

NB15号 0.490 0.399 NB114号 1.017 1.195

NB16 No. 0.747 0.617 NB115 No. 1.106 0.865

NB17 0.669 0.554 NB116 0.609 0.600

NB18 0.770 0.683 NB117 0.931 0.791

NB19 0.490 0.401 NB118 0.660 0.674

NB20 No. 0.556 0.319 NB119 No. 0.884 0.667

NB21 No. 0.879 0.634 NB120 No. 1.253 1.380

NB22号 0.397 0.363 NB121号 0.575 0.588

NB23 0.463 0.402 NB122 0.690 0.630

NB24 No. 0.657 0.572 NB123 0.612 0.536

NB25号 0.824 0.526 NB124号 1.018 0.685

NB27 0.996 0.880 NB125 0.371 0.276

NB28 No. 0.463 0.384 NB126 No. 0.619 0.435

NB29 No. 1.100 0.651 NB127 0.473 0.335

NB30号 0.373 0.328 NB128号 0.679 0.540

NB31号 0.725 0.678 NB129号 0.758 0.621

NB32 No. 0.664 0.422 NB130 No. 0.518 0.390

NB33号 0.464 0.354 NB131号 0.301 0.306

NB34 0.689 0.628 NB132 0.519 0.491

NB35 0.503 0.437 NB133 0.595 0.506

NB36 0.382 0.390 NB134 0.672 0.618

NB37 0.784 0.697 NB135 0.452 0.380

NB38 0.521 0.442 NB136 0.582 0.533

NB39号 0.392 0.331 NB137号 1.068 0.910

NB40 No. 0.473 0.393 NB138 No. 0.570 0.537

NB41 0.448 0.443 NB139 0.631 0.634

NB42 0.365 0.582 NB140 0.316 0.294

NB43 0.679 0.618 NB141 0.685 0.606

NB44 0.316 0.301 NB142 0.499 0.473

NB45号 0.306 0.301 NB143号 0.828 0.737

NB46 0.367 0.352 NB144 0.501 0.533

NB47 No. 0.289 0.237 NB145 0.478 0.448

NB48 No. 0.283 0.258 NB146 No. 0.517 0.537

NB49#0.354 0.317 NB147号0.554 0.486 NB50 No. 0.876 0.903 NB148 No. 0.416 0.357

NB51 0.615 0.617 NB149 0.704 0.515

NB52 No. 0.606 0.462 NB150 No. 0.491 0.357

NB53 0.440 0.365 NB151 0.479 0.403

NB54 0.612 0.458 NB152 0.797 0.619

NB55号 0.837 0.707 NB153号 0.662 0.594

NB56 No. 0.583 0.487 NB154 No. 0.744 0.612

NB57 0.495 0.384 NB155 0.305 0.237

NB58号 0.666 0.559 NB156号 1.027 0.931

NB59 0.888 0.799 NB157 0.628 0.485

NB60 No. 0.682 0.621 NB158 No. 0.320 0.230

NB61 0.433 0.398 NB159 0.414 0.328

NB62 0.783 0.626 NB160 0.538 0.435

NB63 No. 0.404 0.334 NB161 No. 0.282 0.348

NB64 No. 0.515 0.418 NB162 0.484 0.453

NB65 No. 0.982 0.793 NB163 No. 0.341 0.308

NB66 0.364 0.348 NB164 0.552 0.425

NB67 0.518 0.501 NB165 0.631 0.459

NB68 0.469 0.438 NB166 0.480 0.385

NB69 0.496 0.487 NB167 0.774 0.615

NB70 No. 0.536 0.469 NB168 No. 0.552 0.467

NB71 0.430 0.507 NB169 0.493 0.350

NB72 1.482 1.469 NB170 0.384 0.329

NB73 0.611 0.579 NB171 0.473 0.437

NB74 0.534 0.517 NB172 0.299 0.282

NB75 No. 0.522 0.507 NB173 No. 0.257 0.254

NB76 0.448 0.368 NB174 0.324 0.329

NB77 No. 0.500 0.515 NB175 No. 0.176 0.193

NB78 No. 0.425 0.344 NB176 No. 0.258 0.243

NB79 No. 0.739 0.554 NB177 0.358 0.396

NB80 No. 0.504 0.437 NB178 0.646 0.697

NB81 No. 0.684 0.525 NB179 0.486 0.442

NB82 No. 0.787 0.675 NB180 No. 0.507 0.587

NB83 0.998 1.021 NB181 0.207 0.265

NB84号 0.893 0.701 NB182号 0.703 0.629

NB85 No. 0.698 0.580 NB183 0.447 0.370

NB86 No. 0.873 0.958 NB184 No. 0.364 0.399 N B87 No. 1.370 1.241 N B185 No. 0.289 0.261

N B88 0.591 0.493 N B186 0.595 0.452

N B89 0.591 0.488 N B187 0.620 0.643

N B90 0.659 0.641 N B188 0.317 0.329

N B91 0.512 0.454 N B189 0.468 0.480

N B92 0.724 0.634 N B190 0.365 0.339

N B93 1.038 0.854 N B191 0.426 0.397

N B94 0.448 0.455 N B192 0.548 0.520

N B95 No. 0.589 0.542 N B193 No. 1.121 1.128

N B96 0.590 0.592 N B194 0.866 0.804

N B97 1.218 1.009

 N B98 0.560 0.522

 N B99 0.295 0.385

 N B100 0.459 0.476

3. Select 5 polypeptides (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ

ID NO: 5) The patient's serum was detected by ELISA method as the coated and encapsulated ELISA plate. The pathological diagnosis results of the patient are shown in Table 3. Some of the patients were examined by HC2 pathology. The results are as follows:

 Table 3: HPV blood ELISA test patient serum results table

 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID

Serum number pathological data NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 Patient No. 1 CIN III 0.917 0.533 0.723 0.852 0.643 Patient No. 2 CIN II 1.290 0.640 0.714 1.225 1.085 Patient No. 3 CIN II 0.782 0.516 0.295 0.822 0.684 Patient No. 4 CIN III 1.100 0.708 0.676 1.413 0.886 Patient No. 5 Cervical Cancer 0.489 0.344 0.447 0.708 0.536 Patient No. 6 Ovarian Cancer 0.472 0.419 0.447 1.035 0.535 Patient No. 7 Uterine Fibroids 0.909 0.326 0.263 0.844 0.395 Patient No. 8 Cervical Lesion 0.566 0.507 0.492 0.930 0.693 Patient No. 9 CIN III 0.292 0.191 0.184 0.578 0.498 Patient No. 10 CIN III 1.122 0.833 0.768 1.340 1.025 Patient No. 11 Pot 0.466 0.271 0.154 0.401 0.305 Patient No. 12 Cervical Lesion 0.577 0.623 0.499 0.683 0.568 Patient No. 13 Ovarian Teratoma 0.323 0.244 0.206 0.408 0.326 Patient No. 14 CIN II 1.122 0.716 0.789 1.104 0.985 Patient No. 15 Endometrial Cancer 0.366 0.492 0.328 0.341 0.364 Patient No. 16 Cervical Lesion 0.820 0.579 0.395 0.795 0.580 Patient No. 17 uterine fibroids 0.442 0.465 0.283 0.417 0.453 Patient No. 18 ovarian cyst 0.770 0.801 0.660 0.745 1.116 Patient No. 19 Cervical lesions 0.600 0.460 0.299 0.575 0.435 Patient No. 20 Cervical lesions 0.575 0.435 0.334 0.550 0.452 Patient 21 CIN III 0.810 0.469 0.364 0.785 0.722 Patient No. 22 CIN III 0.644 0.550 0.542 0.619 0.742 Patient No. 23 Pot 0.725 0.722 0.676 0.700 0.479 Patient No. 24 CIN II 0.628 0.659 0.391 0.593 0.524 Patient No. 25 Endometritis 0.770 0.332 0.274 0.735 0.359 Patient No. 26 uterine cavity 0.637 0.515 0.491 0.602 0.705 Patient No. 27 Ovarian Cyst 0.467 0.334 0.328 0.432 0.336 Patient No. 28 Ovarian Cyst 0.722 0.547 0.624 0.687 0.434 Patient No. 29 Cervical Lesion 0.621 0.597 0.376 0.586 0.443 Patient No. 30 Ovarian Cyst 0.664 0.763 0.453 0.629 0.496 Patient No. 31 Cervical Malignant tumor 0.600 0.535 0.598 0.565 0.530 Patient 32 cervical cancer 0.841 0.915 0.660 0.806 0.880 Patient 33 CIN II 0.585 0.500 0.519 0.571 0.425 Patient No. 34 endometrial cancer 0.788 0.641 0.514 0.753 0.514 Patient 35 cervical cancer after surgery 0.772 0.438 0.439 0.737 0.471 Human chromosome 36 CIN III 0.661 0.613 0.604 0.704 0.625 1.197 0.805 Patient 37 0.780 1.132 0.898 cancer patients ovarian cyst 38 0.736 0.478 0.387 0.671 0.662 Patient No. 39 CIN III 0.807 0.589 0.842 0.742 0.615 Patient No. 40 CIN III 1.542 1.412 1.553 1.477 1.418

4. Select 3 polypeptides (SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5) as the coated and encapsulated ELISA plate to detect the patient's serum by ELISA, and the patient's pathological diagnosis result As shown in Table 4, the results are below:

 Table 4: HPV blood ELISA test patient serum results table

P240 Cervical flat wet sputum 0.333 0.756 0.753

P255 CIN II 0.378 0.773 0.683

P269 CIN I 0.380 0.567 0.617

P289 Fallopian tube empyema 0.471 1.449 0.747

P302 Adenomyosis 0.238 0.288 0.344

P322 pelvic inflammatory disease 0.255 0.389 0.451

P352 uterine fibroids 0.623 0.741 0.632

P358 uterine fibroids 0.600 0.728 0.607

P408 Left ovarian teratoma 0.303 0.530 0.821

P420 Chronic cervicitis 1.090 1.407 1.575

P421 CIN I - II 0.493 0.708 0.844

P421 CIN1-2 grade 0.382 0.492 0.455

P427 CIN III 0.281 0.671 0.642

After P444 CIN 0.300 0.828 0.633

P449 squamous cell carcinoma 0.200 0.547 0.497

P463 CIN3 grade 0.545 0.610 0.507

P467 CIN3 grade 0.204 0.530 0.480

P471 CIN III 0.514 0.335 0.320

P482 CIN III 0.297 0.724 0.594

P482 CIN III 0.319 0.689 0.522

P483 cervical squamous cell carcinoma 0.289 0.669 0.539

P487 CIN 0.420 0.698 0.796

P488 CIN II 0.321 0.640 0.553

P489 CIN 0.233 0.790 0.887

P490 Cervical lesions 0.135 0.270 0.327

P491 CIN I 0.545 0.358 0.448

P493 Cervical lesions 0.256 0.516 0.434 III. Data analysis

 1. Calculate the average value, standard deviation, and CUTOFF value based on the normal person detection value. The OD values of the 44 normal human sera in Table 1 on the HPVE7 polypeptide were averaged and the standard deviation SD was calculated. The kit selects the normal human mean plus twice the standard deviation when determining the CUTOFF value. The judgment criteria for HPV negative positives are as follows:

 Cutoff is calculated as: cutoff = average OD + 2 * SD standard deviation

 1.1 According to the results of Table 1, HPVE7 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:

4, SEQ ID NO: 5) Statistical analysis of 5 peptides detected in normal human serum results, the results are as follows:

Table 5: HPP E7 5 peptide serum ELISA test data sheet SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 Average 0.292 0.279 0.382 0.301 0.383 Standard deviation 0.098 0.100 0.111 0.076 0.111 cutoff 0.487 0.480 0.604 0.452 0.605

1.2 According to the results of Table 2, two samples of HPVE7 (SEQ ID NO: 3 and SEQ ID NO: 5) were detected by statistical analysis of normal human blood results. The results are as follows:

 Table 6: HPV E7 2 peptide serum ELISA test data sheet

1.3 According to the results of Tables 5 and 6, the comparative analysis showed that the two peptides of SEQ ID NO: 3 and SEQ ID NO: 5 were consistent in the cutoff values obtained in different experiments.

 2. By using the cutoff value, it is judged that the detected patient's serum HPV is negative and positive. If the cutoff value is exceeded, it is considered to be positive, and as long as one peptide is tested positive, HPV infection is considered and compared with the HC2 diagnosis.

 2. 1 291 cases of serum in the tested patients had HC2 diagnosis, and 84 cases had pathological diagnosis. In the pathologically positive group (clinical diagnosis "gold standard"), serum antibody test results were compared with HC2 molecular test results. The ratio (see Table 7) is compared with the HPV test results of this kit as follows: (The positive results in this test include: (i) Any one of the two antigen test results is a positive result, (ii) Both antigen test results are positive results)

 Table 7: According to the results of Table 3, the comparison of serum ELISA results and HC2 diagnosis results for patients

 ( "+" means the test result is positive; "-" means the test result is negative)

Patient No. 14 CIN II + + + + + + Patient No. 15 Endometrial Cancer + +

Patient No. 16 Cervical lesions + + + + + Patient No. 17 Uterine fibroids +

Patient No. 18 Ovarian cyst + + + + + Patient No. 19 Cervical lesions +

Patient No. 20 Cervical lesions + +

Patient 21 CIN III + + + + Patient 22 CIN III + + + + + Patient 23 pelvis + + + + + + Patient 24 CIN II + + +

Patient No. 25 Endometritis + + Patient No. 26 Intrauterine Occupation + + + + +

Patient No. 27 ovarian cyst

Patient No. 28 Ovarian cyst + + + + + Patient No. 29 Cervical lesion + +

Patient 30 ovarian cyst + + + + + patient 31 cervical cancer + + + + +

Patient No. 32 Cervical Cancer + + + + + + Patient No. 33 CIN II + + + +

Patient No. 34 Endometrial Cancer + + + + + + Patient No. 35 Cervical Cancer Postoperative + + + Patient No. 36 CIN III + + + + + + Patient No. 37 Cervical Cancer + + + + + + Patient No. 38 Ovarian Cyst + + + + Patient No. 39 CIN III + + + + + + Patient No. 40 CIN III + + + + + + Table 8: According to the results of Table 4, the comparison of serum ELISA results and HC2 diagnosis results for patients

P269 CIN I + + +

P289 Fallopian tube empyema + + + +

P302 adenomyosis

 P322 pelvic inflammatory disease

 P352 uterine fibroids + + +

P358 uterine fibroids + + +

P408 left ovarian teratoma + +

P420 Chronic cervicitis + + +

P467 CIN3 + +

P482 CIN III + +

P483 cervical squamous cell carcinoma + +

P487 CIN + + +

P488 CIN II + + +

P489 CIN + + +

P490 Cervical lesions

 P491 CIN I + +

P493 Cervical lesions + - + + The HC2 results of the results of Table 7 and the results of blood ELISA were statistically analyzed. The results are as follows:

 Table 9: HC2 results and blood ELISA detection accuracy rate and proportion comparison table

 Other gynecological diseases are mainly pelvic inflammatory disease, ovarian swelling, uterine fibroids and other gynecological diseases. The HC2 results of the results in Table 8 and the blood ELISA results were statistically analyzed. The results are as follows:

 Table 10: HC2 results and blood ELISA detection accuracy rate and proportion comparison table Pathological diagnosis results Number of people HC2 detection SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 positive positive positive positive

CIN I 4 3 2 3 4

CIN II - III 7 4 0 7 7 Cervical cancer 1 1 0 1 1 Cervical lesion 2 1 0 1 1 Other gynecological diseases

 Other gynecological diseases are mainly pelvic inflammatory disease, ovarian swelling, uterine fibroids and other gynecological diseases.

2. 2 There are 10 cases of serum in the patient's serum without HC2 test report, but the pathological report is CIN and cervical cancer. The kit detects HPV results. The comparison of the number of patients and the positive rate of blood test is shown in the table.

 Table 11: Summary of HPV blood ELISA results for patients without HC2 diagnosis, but with pathological findings

 ( "+" means the test result is positive; "-" means the test result is negative)

Table 12: Number of patients with CIN, cervical cancer and cervical lesions without blood HC2

2. 3 The results of blood ELISA test results and positive proportions of all patients with CIN II and above and cervical cancer are shown in Table 13.

 Table 13: Proportion of the number and accuracy of blood ELISA for all patients with CIN, cervical cancer and cervical lesions

The above results indicate that the antigen peptides SEQ ID NO: 2, 3, 5 are more sensitive and accurate for CIN II detection; and the antigen peptides SEQ ID NO: 3, 4, 5 are more sensitive and accurate for CIN III and cervical cancer detection. excellent. The above polypeptide of the present invention is selected by the inventors and verified in the patient sample and the normal human sample, and the GST-HPV16-E7 and GST-HPV118E7 full-length fusion proteins (SEQ ID NO: 6 respectively) are directly used. The sequence shown in SEQ ID NO: 7) is not effective, please refer to the following experimental data.

 4. The experimental materials and ELISA methods are the same as the first point above, and the GST-HPV16-E7 and GST-HPV118E7 full-length fusion proteins are also used together with the detection of the polypeptide (SEQ ID NO: 3, SEQ ID NO: 5). As a detection antigen, when the GST-HPV16-E7 and GST-HPV18-E7 full-length fusion proteins were diluted with PBS, the concentration was 2 ug/ml, and the results were as follows:

 1. According to the above experimental steps, some patient serum and ordinary human serum are detected. The test results and patient pathology report are as follows:

 Table 14 Peptide detection and full-length fusion protein test results

P319 pelvic inflammatory disease 0.452 0.427 1.579 1.015

P320 pelvic inflammatory disease 0.498 0.427 0.275 0.525

P321 pelvic inflammatory disease 0.515 0.662 0.504 0.497

P322 pelvic inflammatory disease 0.393 0.457 0.516 0.506

P323 Nodules 0.186 0.663 0.420 0.335

P451 No. Pot 0.868 0.595 0.452

P452 uterine cavity occupancy 0.891 0.601 0.485

P453 CIN 1.194 0.672 0.425

P454 Cervical malignancy 0.625 0.687 0.334

P455 Cervical cancer 1.076 0.848 0.976

P456 CIN II 0.977 0.820 0.570

P457 Endometrial cancer 1.326 0.925 0.630

P458 Cervical cancer surgery 0.986 0.972 0.547

P459 Cervical cancer surgery 0.857 1.205 0.645

P460 CIN III 0.483 0.754 0.450

P461 Fallopian tube cancer 0.937 0.855 0.543

P462 Cervical cancer 0.907 0.789 0.525

P463 Cervical Cancer 1.072 0.958 0.532

P464 Cervical lesions 0.893 0.961 0.618 Normal group:

 NA1 0.656 0.483 0.255 0.282

NA2 0.461 0.998 0.119 0.215

NA3 0.367 0.580 0.210 0.233

NA5 0.427 0.776 0.192 0.191

NA11 0.370 0.758 0.198 0.333

NA12 0.307 0.665 0.250 0.281

NA16 0.328 0.641 0.131 0.225

NA18 0.445 0.622 0.260 0.293

NC24 0.376 0.791 0.290 0.421

NC31 0.308 0.319 0.390 0.485

NC36 0.335 0.492 0.197 0.464

NC38 0.356 0.452 0.490 0.588

NC40 0.346 0.432 0.150 0.331

NC41 0.530 0.882 1.024 1.134

NC79 No. 0.285 0.380 0.329 0.455

NC101 0.389 0.577 0.307 0.422

NC102 No. 0.405 1.057 0.346 0.324 V. Experimental analysis

 1. Compare the detection results using the full-length protein with the results of the previous detection of the HPV polypeptides SEQ ID: 3 and SEQ ID: 5, for example, in the table below, HPV16-E7 protein, HPV18-E7 protein, antigen The cutoff values of the peptide SEQ ID: 3 and the antigen peptide SEQ ID: 5 are: OD values of 0.601 and 0.603, respectively, in the detection of the polypeptide, as long as the antigen peptide SEQ ID: 3 and the antigen peptide SEQ ID: 5 are detected. If it is positive, the result is considered positive.

 Table 15 Analysis of peptide detection and full-length fusion protein detection results

P322 pelvic inflammatory disease

 P323 uterine fibroids +

 P451 Pot + +

 P452 uterine cavity occupying + +

 P453 CIN + + +

P454 Cervical malignancy + + +

P455 Cervical Cancer + + +

P456 CIN II + + +

P457 Endometrial cancer + + +

P458 Cervical Cancer Postoperative + + +

P459 Cervical Cancer Postoperative + + +

P460 CIN III + +

P461 Fallopian tube cancer + + +

P462 Cervical Cancer + + +

P463 Cervical Cancer + + +

P464 Cervical lesions + + +

 Normal group:

 NA1 +

 NA2 +

 NA3 +

 NA5 +

 NA11 +

 NA12 +

 NA16 +

 NA18 +

 NC24 +

 NC31

 NC36

 NC38

 NC40

 NC79

 NC101 +

NC102 + Summary: HPV16-E7, HPV18-E7 full-length fusion protein in the detection of CIN, cervical cancer patients have a certain positive patient missed detection, sensitivity is poorer than peptide detection. There is a certain false positive rate in the detection of general gynecological inflammatory patients and the general population, and the sensitivity is slightly worse than the peptide detection. Therefore, HPV 16-E7 and HPV 18-E7 full-length proteins are not very suitable for detecting CIN, cervical cancer, and the like. Example 2 Specificity verification

 In this example, in order to further verify whether the SEQ ID No.: 1-5 antigen peptide has a specific epitope of HPV, the ELISA method is used to detect the full-length HPV 16-E7 protein, or HPV 18-E7 protein. The monoclonal antibody obtained as an immunizing antigen was subjected to specific binding test with each antigen peptide. Among them, each monoclonal antibody is a monoclonal antibody which specifically binds to the full-length 16-E7 or 18-E7 antigen.

 As a result, it was found that the antigen peptides of SEQ ID NOS: 1-5 can bind to various monoclonal antibodies. This result suggests that the antigenic peptide of the present invention has an epitope capable of specifically binding to a human papillomavirus antibody (see the table below). Also. SEQ ID No.: 1-5 also does not bind to other unrelated antibodies (including antibodies against other viral proteins), suggesting that the antigenic peptide of the present invention is cross-reactive.

 Table 16 Antigenic peptide epitope analysis

Therefore, the antigenic peptide of the present invention can be used not only for detecting (especially early detection) cancer caused by HPV infection and HPV infection, but also for preparing a prophylactic or therapeutic pharmaceutical composition (including a vaccine composition), thereby Effectively induces the body to produce a specific immune response against human papillomavirus HPV oncoprotein, thereby preventing cancer or treating cancer. discuss

The blood enzyme-labeled immunoassay (ELISA) of the present invention provides an antigen-antibody detection method with high sensitivity, high specificity and high stability based on the principle of immunobiochemistry. The patient develops specific antibody IgM, IgA or IgG molecules in serum and oral saliva after infection by the virus. Antibody-specific anti-HPV antibody molecules in serum or saliva were detected by enzyme-labeled immunoassay ELISA. The invention predicts the antigenicity by bioinformatics analysis through the analysis of HPV virus high-risk HPV16 and HPV18 gene sequences, and uses experimental methods to screen and prove the significance of clinical application. By preparing a plurality of high-purity antigenic polypeptides, and repeatedly screening and verifying the prepared antigens by the serum of the domestic population and the serum of the infected person, the target antigen HPV16-1 with high sensitivity, high specificity and high stability was obtained. HPV 16-2, HPV 16-3, HPV18- HPV18-2. It has also been demonstrated that the binding of these polypeptide antigens to the surface of a solid phase carrier maintains its immunoreactive activity. At the time of measurement, the test specimen is allowed to react with the antigen on the surface of the solid phase carrier. The antigen, the antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the amount of the enzyme bound to the immobilized carrier is proportional to the amount of the test substance in the sample. After adding the substrate of the enzyme reaction, it is then detected by color development, so it can be qualitatively or quantitatively analyzed according to the depth of the color reaction. Compared with DNA detection (HC2, CareHPV), the blood ELISA detection kit of the invention has uniform sampling, non-aggressiveness, quicker operation and simple operation; low cost, easy promotion, and more suitable for the Chinese market; False positives and false negatives greatly improve the accuracy and accuracy of the test results; compared with traditional Pap smear, acetic acid stained VIA, colposcopy, more accurate, easy and fast. ELISA detection method through various parameters and experimental procedures The optimization can be used as a tool for large-scale screening for early screening of cervical cancer.

 In summary, the antigen for detecting an anti-human papillomavirus antibody of the present invention can detect HPV antibodies in blood or saliva with high sensitivity, high specificity and high stability, thereby accurately diagnosing cervical intraepithelial neoplasia for a doctor. And cervical cancer to provide the basis, and then timely treatment to prevent cancer, or cancer spread, timely relief of patient suffering, the immunoassay kit made of it can quickly and accurately detect HPV antibodies in blood or saliva, Suitable for large-scale promotion of applications.

 In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and changes can be made without departing from the spirit and scope of the invention. Accordingly, the description is to be considered as illustrative and not restrictive.

Claims

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A human papillomavirus antigen peptide, characterized in that the antigen peptide is derived from an E7 protein of human papillomavirus, and the antigen peptide is 5 to 50 amino acids in length, and the antigen peptide is An antibody that binds to human papillomavirus.

 The antigenic peptide according to claim 1, wherein the m+2th position, the m+3th position, the m+5th position, and the m+6th position of the antigen peptide are Asp, Ser, and Glu. The Glu and/or the m+8-m+12 amino acid are in turn Asp, Glu, Ile, Asp and Gly, wherein m≥0JLm is an integer.

 The antigen peptide according to claim 1, wherein the antigen peptide has a structure as shown in Formula I

 [XaaO]-[Xaal]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6] (I)

 In the formula,

 XaaO is a peptide consisting of none or 1-5 amino acids;

 Xaal is DS;

 Xaa2 is selected from S or E;

 Xaa3 is EE;

 Xaa4 is selected from E or N;

 Xaa5 is DEIDG;

 Xaa6 is a peptide consisting of no or a length of 1 to 30 amino acids.

 The antigenic peptide according to claim 3, wherein the peptide sequence of XaaO and/or Xaa6 is an E7 protein sequence derived from HPV.

 The antigenic peptide according to claim 4, wherein the HPV virus comprises HPV16, HPV18,

HPV3 HPV33, HPV35, HPV39, HPV45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 68.

 The antigenic peptide according to claim 1, wherein the antigen peptide has a structure as shown in Formula II

 [YaaO]-[Yaal]-[Yaa2]-[Yaa3]-[Yaa4]-[Yaa5] (II)

 In the formula,

 YaaO is a peptide consisting of no, or 1-30 amino acids;

 Yaal is DS;

 Yaa2 is selected from S or E;

 Yaa3 is EE;

 Yaa4 is selected from E or N;

 Yaa5 is a peptide consisting of no, or 1-30 amino acids in length.

 The antigenic peptide according to claim 6, wherein the YaaO is a peptide consisting of 5-25 amino acids and Yaa5 is a peptide consisting of 1-10 amino acids; or

 The YaaO is a peptide consisting of 1-5 amino acids and Yaa5 is a peptide consisting of 10-25 amino acids;

 The peptide sequence of and/or YaaO and/or Yaa5 is the E7 protein sequence derived from HPV.

 The antigenic peptide according to claim 1, wherein the antigenic peptide is selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 5 or a combination thereof.

The antigenic peptide according to claim 1, wherein the antigenic peptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of any of SEQ ID NOS: 1-5;

 (b) a polypeptide having > 80% homology to the amino acid sequence shown in any one of SEQ ID ΝΟ: 1-5, and having the ability to bind to an antibody against human papillomavirus;

 (c) Derivation of the amino acid sequence of any one of SEQ ID ΝΟ: 1-5 by substitution, deletion or addition of 1-5 amino acid residues, and retaining the ability to bind to antibodies against human papillomavirus Peptide.

 A fusion protein, characterized in that the fusion protein comprises the antigen peptide of any one of claims 1-9 and an optional tag sequence.

 An isolated nucleic acid molecule, which encodes the antigen peptide of any one of claims 1 to 9 or the fusion protein of claim 10.

 12. A composition comprising:

 (a) The human papillomavirus antigen peptide of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, or the fusion protein of claim 10;

 (b) a pharmaceutically acceptable carrier or excipient.

 13. The composition of claim 12, wherein the composition comprises a pharmaceutical composition and a vaccine composition.

 Use of the human papillomavirus antigen peptide according to any one of claims 1 to 9, characterized in that it is used for preparing a reagent or a kit for detecting HPV; or

 For the preparation of a medicament for the prevention or treatment of human papillomavirus infection; or

 For detection of anti-human papillomavirus antibodies.

 An immunoassay kit, comprising the human papillomavirus antigen peptide according to any one of claims 1-9.