CN1775802A - Human papillomavirus immunogenic polypeptide, and its preparing method and use - Google Patents
Human papillomavirus immunogenic polypeptide, and its preparing method and use Download PDFInfo
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- CN1775802A CN1775802A CN 200410091057 CN200410091057A CN1775802A CN 1775802 A CN1775802 A CN 1775802A CN 200410091057 CN200410091057 CN 200410091057 CN 200410091057 A CN200410091057 A CN 200410091057A CN 1775802 A CN1775802 A CN 1775802A
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Abstract
The invention relates to the manufacturing method for human kind papillomavirus immunogenicity polypeptide and the application, which belongs to biology technology. The E6 and E7 polypeptide sequence of human kind papillomavirus HPV16 and 18 contain all the amino acid sequence in the sequence table from SEQ ID NO.1 to SEQ ID NO.33. The polypeptide could be used to make medicine for curing HPV infect and venereal diseases caused by HPV infect, and benign tumor or malignant tumor.
Description
Technical field
The present invention relates to human papillomavirus immunogenic polypeptide, its preparation method and application, especially refer to immunogenic by having of obtaining of immunization screening in bioinformatic analysis, inhibition competition receptor-ligand affinity method and external and the body, can and HLA-A1, A2, A3, the E6 of A11 and A24 bonded human papillomavirus HPV16 and 18 and E7 peptide sequence, the preparation method of these polypeptide with and be used to prepare prevention and treatment HPV infects and by the application that HPV infects the medicine of the venereal disease that causes and good, malignant tumour, belong to biological technical field.
Background technology
Papilloma virus (Papillomavirus) can infect multiple biology, and it has species specificity.The animal teat tumor virus can not infect the mankind, and (Human Papillomavirus HPV) can infection animal for same human papillomavirus.HPV belongs to the no tunicle virus of double stranded DNA (DNA), the tissue specificity of having a liking for is arranged, the epithelium of easy infection human body skin and mucous membrane, often cause men and women's anus edeitis, epithelial proliferation, and then develop into innocent tumour, as pointed condyloma (claiming venereal wart condyloma acuminata again) or malignant tumour, as cervical cancer.HPV has a different hypotype surplus in the of 130, and the HPV that finds in the generaI investigation of anus sexual organ has 23 kinds approximately, and wherein some usually causes benign lesion, is called low risk HPV (low-risk HPV), as HPV11 and HPV6.Some usually causes malignant change, is called high risk HPV (high-risk HPV), as HPV16 and 18.The carcinoma vulvae patient HPV16 of the cervical cancer of 85%-90% and 35%-50% and the HPV18 positive (W.J.vanDriel, et al, Eur J Cancer, 35:946-952,1999).Usually after HPV infected 10 years, pilosity was born in women's breeding time in the generation of cervical cancer.There is report to claim about 25%-33% cervical cancer to betide behind the pointed condyloma 25 years.Cervical cancer is second common cause of global women because of tumor mortality.Can find HPV DNA in the focus of cervical cancer patient above 94 percent, it is relevant with cervical cancer to illustrate that HPV infects.The genome of HPV contains 8000 base pairs, can express two genoids: early gene (E) participates in dna replication dna, and protein is transcribed and cell transformation (transformation), and late period, (L) gene was responsible for instructing synthetic viral capsid proteins.The albumen of E6 genetic expression can with the p53 protein binding, the albumen of E7 genetic expression can with retinoblastoma (Rb) protein binding.P53 and Rb albumen are tumor suppressor proteins, and under normal circumstances they participate in the division and the hyperplasia of control cell.E6 and E7 albumen with make its inactivation after it combines, activating cells is transcribed mechanism, makes infected cell enter cell generation cycle, hyperplasia transforms and canceration.Because it is the most common that E6 in the early gene and E7 are expressed in the tumour that HPV causes, can promote the generation of tumour, and find to contain the restricted T cell antigen of MHC I class determinant (MHC class I restricted Tcell epitopes) in HPV E6 and the E7 albumen, therefore, this established a kind of design with E6 in HPV 16 and 18 and E7 antigenic determinant be content, be used to prevent and treat that HPV infects and the basis of the optimum or malignant tumour vaccine that causes.
Immunity system is organism opposing and the securing system of removing invasion pathogenic agent and mutant cell, and it can be divided into immunity system (antibody) and cell immune system.A large amount of bases and clinical immunology research have confirmed that HPV antigen can induce the HPV specific cell immunoreaction, and cellular immunization is playing an important role in the pathology due to controlling HPV.Clinical observation finds that most of women can fall virus sweep by autoimmunization after infecting HPV 9-15 month, even some patient's Genital warts can spontaneous regression.Intracutaneous knurl on severe uterine cervix and the vulva (CIN/VIN) patient's T lymphocyte is to HPV L1 for Cervical Intraepithelial neoplasia, VulvarIntraepithelial neoplasia, E2, and E4 and E7 albumen stimulate can produce proliferation response.In mouse test, with the tumour cell of expressing HPV E7 and T cell co-stimulatory molecules (costimulatorymolecule) B7, or the tumour cell of transfection HPV16 complete genome, or can induce with the vaccinia virus member that contains total length HPV E7cDNA and to produce HPV E7 specificity T killer cell.HPV E7 polypeptide associating adjuvant (IFA) immune mouse can be protected the attack of mouse opposing HPV E7 positive tumor subsequently.Give and to have set up the HPV tumour and do not have adopting property of the nude mice input HPV16 E7 polypeptide vaccine institute inductive cytotoxic T cell of T lymphocyte immunity ability that (Cytotoxic T Lymphocyte CTL) can remove the HPV tumour .HPV that has set up and can induce the discovery of HPV specific cell immunoreaction further to be confirmed in HPV16 positive uterine cervix atypical hyperplasia and cervical cancer patient.The lymphocyte of patient's peripheral blood and lymphoglandula shows HPV E7 specific killing function with after being combined with the antigen presenting cell repetitious stimulation of HPV E7 polypeptide.Virus-positive is also found in research, and the negative patient's of CIN cell killing function ratio CIN positive patients is strong.Be reported in and have HPV specificity memory T killer cell in some HPV infected patient body.Other has report HPV specificity T killer cell to be more common in early days in the late period of pathology, these find that prompting HPV specific killing cell function affects the generation and the development of tumour. the report of the HPV16 E7 of American South University of California polypeptide vaccine first phase clinical test results, intracutaneous knurl patient uterine cervix scraping blade proof virus after the 4th vaccinate is eliminated on 66% HPV16 positive height uterine cervix and the vulva.The part or all of extinction rate of focus reaches 50%.The immune response of HPV E6 and E7 antigen induction may be able to be controlled the virus movable (M.R.Hilleman, J.Clin.Virology, 19:79-90,2000) of HPV acute infection period.
In sum, HPV antigen can induce the host to produce HPV antigen-specific cell immune response, and this cell immune response has the opposing virus infection, and prophylaxis of tumours takes place, and suppresses tumor growth and the effect that impels tumor regression.The application of HPV polypeptide vaccine can be induced the cell immune response of this protectiveness on one's own initiative, the immunological competence of enhancing body, infectious diseases and tumour that prevention HPV infects and brings out.
Treatment for the HPV infected patient mainly contains medicine and Physiotherapy two classes at present.Pharmacological agent has Pu Dafeiren fat, 5 FU 5 fluorouracil, bleomycin, Interferon, rabbit etc.Physiotherapy has that electricity irons, laser therapy, cryosurgery and surgical resection.Present pharmacological agent does not have specificity, and systemic administration has significant side effects, local application's inconvenience.Physiotherapy is not suitable for HPV and infects the early metaphase patient, only be applicable to that epithelial dysplasia and cancerous swelling have taken place patient after, and can bring misery and adverse consequences to patient.Uterine neck must be taked part or all of uterine cervix resection operation after developing into the property invaded cancer usually, and this is unfavorable for women's fertility and other normal physiological function.Under present therapy, pointed condyloma has the chance recurrence of 20%-50%, and the mortality ratio of the property invaded cervical cancer is up to 60%.This shows that HPV infects does not also have a kind of special effective therapy at present, it is imperative to research and develop a kind of special effective therapy.
At least need ten years in latent period of developing into cervical cancer after HPV infects, this is very suitable for using a kind of prevention and therapeutic vaccine is controlled the HPV prevention and treated the venereal disease that is caused by HPV, as pointed condyloma and men and women's genitalia cancer etc.Generally acknowledged cell and the humoral immune reaction that HPV antigen can be induced anti-HPV, people generally believe that the HPV vaccine is the venereal disease that prevention at present and treatment are caused by HPV, one of pointed condyloma and the most promising therapy of men and women's genitalia cancer.Development HPV vaccine is one of main direction of current HPV infection and associated diseases prevention and control field thereof.Because HPV can not vitro culture, so be not suitable for developing traditional virus vaccines of attenuation or deactivation.The HPV vaccine of using in clinical trial can reduce DNA, protein or polypeptide three class vaccines basically at present.
The first kind is a dna vaccination, and it belongs to the category of gene therapy.The advantage of dna vaccination be can be in vivo antigen expressed routinely.Antigen presentation under this in addition antigenic expression and the natural situation is approximate, therefore can induce intravital immunity system to produce the immune response of protectiveness effectively enduringly.The dna vaccination that has these two big advantages in theory is that ancient vaccine has injected new vitality, has brought great inspiration once for immunologist and physician.Yet, since the safety problem that in clinical trial, occurs of gene therapy with and in vivo mechanism of action complexity, FDA carries out more careful attitude for the clinical trial of gene therapy, make gene therapy make slow progress in the U.S. and even western countries, this has influence on the development of HPVDNA vaccine equally.
Second class is a protein vaccine, and the protein that is used for vaccine can obtain with gene engineering research, or separates the acquisition of purifying in biological fluid.The advantage of protein vaccine is to comprise whole or most of antigens, and this meets the principle of design of vaccine.But produce the difficulty that protein vaccine has protein source.Separating and purifying protein matter be it is generally acknowledged dangerously in the biological fluid, does not advocate, because it may cause the propagation in latent infection source.It is the main means of producing protein vaccine at present that gene engineering research is produced recombinant protein, but the gene engineering research of present horizontal can not the bigger protein of High-efficient Production molecular weight.Comprise two HPV hypotypes as producing, as 16,18 and each hypotype contain two kinds of albumen, as E6 and E7, totally four kinds of proteinic vaccines, also there are many technical barriers at present in gene engineering research, does not see the report of any this respect so far at home and abroad as yet.Units such as Merk company and GlaxoSlthKline company with the HPV capsid protein L 1 or/and the L2 assembling assembly virus-like particle (Virus-likeParticles is VLPs) as vaccine.VLPs is similar to the HPV virion on form, but does not contain HPV DNA, so still belong to protein vaccine or composition vaccine.HPV VLPs enters I respectively, the II clinical trial phase, and anti-HPV antibody titers is than not vaccinated HPV patient high 40 times (Nature Medicine) in HPV patient's body of Preliminary report inoculation HPV VLPs vaccine.Present studies show that the body opposing HPV be pathogenic mainly depend on cellular immune function, and the cellular immunization merit is mainly induced by HPV E6 and E7.The applicable object of HPV VLPs vaccine and provide protection still remain further to be observed.
The 3rd class is a polypeptide vaccine, but polypeptide chemosynthesis or extract or utilize gene engineering method both protokaryon or eukaryotic expression system were expressed and obtained from cytolemma with biochemical process.The maximum characteristics of polypeptide vaccine are safety, and source (synthesizing) is easy.Because molecular weight is little, same dosage HPV vaccine can comprise the polypeptide of more effective antigenic determinants.Existing enough clinical preceding and clinical trials show that the HPV polypeptide vaccine is safe and effective.The HPV16E7 of American South University of California polypeptide vaccine first phase clinical test results shows safe and effective (real certainly curative effect is judged the III clinical trial phase that awaits) as previously mentioned, but the HPV of University of Southern California polypeptide vaccine only comprises the E7 polypeptide of the restricted HPV16 of HLA-A2.Known HLA-A2 accounts for total crowd's 50%, and HPV16 infects and also only accounts for 50% of about cervical cancer, so the E7 polypeptide vaccine of the HPV16 of University of Southern California only can cover 25% HPV infection colony.In fact, all there is the problem of same fraction of coverage in all the HPV vaccines in clinical trial at present, because most HPV vaccine is only at a kind of HPV hypotype and a kind of target antigen.In recent years existing people attempts a kind of target antigen at two kinds of HPV hypotypes, or a kind of HPV vaccine of two kinds of target antigens of HPV hypotype.These HPV vaccine effective objects have nothing in common with each other, and they are mainly respectively at HPV16,18,11 and 6 virus subtypes, but do not have or the HPV vaccine is seldom arranged at other HPV of two or more different shaped.With regard to antigen, they also respectively have selection.It is antigenic having with early gene E6 or E7 product, and it is antigenic also having with late gene L1 or L2 product.Equally, do not have or seldom have the HPV vaccine to comprise two or more different antigen.
Up to the present, American-European nearly altogether nearly ten HPV vaccines are in the first phase or the second phase of clinical trial; China does not still have the report of carrying out the clinical trial of HPV vaccine.
Summary of the invention
First purpose of the present invention provides energy and HLA-A1, A2, A3, A11 and A24 bonded human papillomavirus immunogenic polypeptide.
Second purpose of the present invention provides the preparation method of human papillomavirus immunogenic polypeptide.
The 3rd purpose of the present invention provides that human immunity originality polypeptide is used to prepare prevention and treatment HPV infects and infected the application of the medicine of the venereal disease that causes and good, malignant tumour by HPV.
Last purpose of the present invention provides the men and women's venereal disease, anus, vulva, uterine neck and the penis innocent and malignant tumour that cause are infected in a kind of prevention and treatment HPV infection and prevention by HPV new generation vaccine.
For achieving the above object, the present invention is by the following technical solutions:
Human papillomavirus immunogenic polypeptide, it has the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.33 in the sequence table.The peptide sequence that uses among the present invention is 13-22 amino acid, and each polypeptide includes a plurality of epitopes, and each epi-position is made of 7-10 amino acid, all can independence or be combined into human papillomavirus's polypeptide vaccine.
| SEQ ID NO：1 | MHQKRTAMFQDPQERPR | HPV 16-E6，1-17 |
| SEQ ID NO：2 | FQDPQERPRKLPQLCTELQ | HPV 16-E6，9-27 |
| SEQ ID NO：3 | KQQLLRREVYDFAFRDL | HPV 16-E6，41-57 |
| SEQ ID NO：4 | LCIVYRDGNPYAVC | HPV 16-E6，57-70 |
| SEQ ID NO：5 | SKI SEYRHYCYSLY | HPV 16-E6，78-91 |
| SEQ ID NO：6 | SEYRHYCYSLYGTTLEQQYNK | HPV 16-E6，81-101 |
| SEQ ID NO：7 | MHGDTPTLHEYMLDLQ | HPV16-E7，1-16 |
| SEQ ID NO：8 | TLHEYMLDLQPETTDLYCY | HPV16-E7，7-25 |
| SEQ ID NO：9 | YMLDLQPETTDLYCYEQLND | HPV16-E7，11-30 |
| SEQ ID NO：10 | DEIDGPAGQAEPDRAHYNIV | HPV16-E7，36-55 |
| SEQ ID NO：11 | GQAEPDRAHYNIVTFC | HPV16-E7，43-58 |
| SEQ ID NO：12 | RAHYNIVTFCCKCDST | HPV16-E7，49-64 |
| SEQ ID NO：13 | STHVDIRTLEDLLMGTLGIV | HPV16-E7，71-91 |
| SEQ ID NO：14 | DIRTLEDLLMGTLGIVCPI | HPV16-E7，75-93 |
| SEQ ID NO：15 | LLMGTLGIVCPICSQKP | HPV16-E7，82-98 |
| SEQ ID NO：16 | TLGIVCPICSQKP | HPV16-E7，86-98 |
| SEQ ID NO：17 | FEDPTRR PYKLPDLCTEL | HPV18-E6，4-21 |
| SEQ ID NO：18 | YKLPDLCTELNTSLQDI | HPV18-E6，12-28 |
| SEQ ID NO：19 | PDLCTE LNTSLQDIEITCVYC | HPV18-E6，15-35 |
| SEQ ID NO：20 | TCVYCKTVLELTEVFEFAFK | HPV18-E6，31-50 |
| SEQ ID NO：21 | EVFEFAFKDLFVVY | HPV18-E6，43-56 |
| SEQ ID NO：22 | CHKCIDFYSRIREL | HPV18-E6，65-78 |
| SEQ ID NO：23 | YSDSVYGDTLEKLTNTGL | HPV18-E6，81-98 |
| SEQ ID NO：24 | TLEKLTNTGLYNLLIRCL | HPV18-E6，89-106 |
| SEQ ID NO：25 | LTNTGLYNLLIRCLRCQKPL | HPV18-E6，93-112 |
| SEQ ID NO：26 | MHGPKATLQDIVLHLEPQNE | HPV18-E7，1-20 |
| SEQ ID NO：27 | KATLQDIVLHLEPQNE IPVDL | HPV18-E7，5-25 |
| SEQ ID NO：28 | LHLEPQNEIPVDLLCHEQ | HPV18-E7，13-30 |
| SEQ ID NO：29 | DSEEENDEIDGVNHQHLPAR | HPV18-E7，33-52 |
| SEQ ID NO：30 | DGVNHQHLP ARRAEP | HPV18-E7，42-56 |
| SEQ ID NO：31 | RAEPQRHTMLCMCCKCEARIEL | HPV18-E7，53-74 |
| SEQ ID NO：32 | DDLRAFQQLFLNTLSFVCPW | HPV18-E7，81-100 |
| SEQ ID NO：33 | QQLF LNTLSFVCPWCASQQ | HPV18-E7，87-105 |
The preparation method of human papillomavirus immunogenic polypeptide, this method may further comprise the steps:
(1) access human papillomavirus 16 from protein library, 18-E6 and E7 protein sequence are with information biology (Bioinformatics) analyses and prediction HLA-A1, A2, A3, the restricted antigenic determinant of A11 and A24, set up HPV16,18-E6 and E7 polypeptide libraries (Peptide Library);
(2) filter out and HLA-A1 from this polypeptide libraries with the affine method of inhibition competition receptor-ligand, A2, A3, A11 and A24 have the polypeptide of binding ability;
(3) further filter out with external immunization and have immunogenic polypeptide;
(4) further screen with immunization in the body and confirmed that these immunogenic polypeptides induce HPV16, the ability of 18-E6 and E7 polypeptid specificity t cell immune response in vivo.
External immunoscreening is a lymphocyte of using HPV polypeptide repetitious stimulation normal people under specific cell culture condition, can stimulate and induce normal people's lymphocyte to produce the immunoreactive polypeptide of HPV polypeptid specificity and be called immunogenic polypeptide, have possibility as vaccine.For the immunogenicity of the polypeptide that confirms to be gone out by external immunoscreening, these polypeptide are used to immune animal.The present invention distinguishes immune Balb/c with polypeptide, C57 and C3H mouse, carry out booster immunization after two weeks one time, the spleen lymphocyte of separation test mouse around the, detect the HPV polypeptid specificity t cell immune response of mouse with immunoenzyme connection spot technology (ELISPOT), and with 51Cr method for releasing analysis of cells toxic T lymphocyte killing ability.The result shows that the HPV polypeptide that the present invention filters out can successfully induce the immune response of HPV polypeptid specificity T cytoprotective in vivo.
Disclosed by the invention have immunogenic human papillomavirus's antigenic peptide and have following application:
One, human papillomavirus immunogenic polypeptide is used to prepare prevention and treats the application that the human papillomavirus infects the vaccine of caused infectious diseases and neoplastic disease.Described infectious diseases is human parillomarvirus infections venereal disease.Described neoplastic disease comprise the human papillomavirus infect due to uterine cervix, vulva, anus, penis and oropharynx proliferative lesion, innocent tumour, precancerous lesion and cancer thereof.
Two, human papillomavirus immunogenic polypeptide is used to prepare the application that the detection human papillomavirus infects the reagent of caused infectious diseases and neoplastic disease.
At problems such as existing HPV vaccine fraction of coverage deficiencies, the present invention has designed a kind of novel HPV vaccine, and this vaccine can cover the main HPV hypotype-16 and 18 hypotypes of bringing out cervical cancer; Can induce the cellular immune function that HPV patient is played main provide protection; Vaccine can be applied to the colony (HLA-A1, A2, A3, A11 and A24) of a plurality of HLA hypotypes; The activeconstituents of vaccine is polypeptide, and is safe in utilization, is convenient to produce and quality-guarantee.
Known cervical cancer is caused by HPV16 and 18 that mainly the inducing cell immunologic function mainly is E6 and the E7 antigen of HPV, so our vaccine has comprised HPV16 and 18 2 kinds of hypotypes and E6 and two kinds of antigens of E7.
Human papillomavirus's prevention and therapeutic vaccine, the activeconstituents in this vaccine is made up of the human papillomavirus immunogenic polypeptide shown in sequence table SEQ ID No.1 to the SEQ ID No.33.
This vaccine also comprises adjuvant or other complementary polypeptide.
The material of energy enhancing body immunne response is called adjuvant.After adjuvant and antigenic substance (as the polypeptide in this patent) inject body, form the antigen storage vault, make antigen slowly be released into immunity system, the antigenic action time lengthening in the part; Can stimulate body to form local inflammation or granuloma, the chance that enhancement antigen contacts with immunocyte; Can produce cytokine by the immune stimulatory cell, enlarge by the inductive immune response of antigen institute.The described adjuvant of this patent is inorganic adjuvant, and as aluminium hydroxide, organic adjuvant is as IFA (Montanide ISA-51), bacterium adjuvant, as BCG, Nuclec acid adjuvants, as CpG, or the cytokine adjuvant, as IL2, IFN α, IFN γ, GM-CSF etc., but be not limited to above-mentioned adjuvant.
Described complementary polypeptide is the restricted polypeptide of MHC II.Depend on the help of t helper cell when the T killer cell is implemented its killing ability in vivo.The restricted polypeptide of MHC II can activate t helper cell.Therefore, in the HPV polypeptide vaccine, add immunoprophylaxis and the therapeutic action that the restricted polypeptide of MHC II can strengthen the HPV polypeptide vaccine.
This vaccine can be made into preparation, comprises HPV immunogenic polypeptide, solvent and adjuvant.White powder HPV polypeptide for example; A kind of solvent as stroke-physiological saline solution, but is not limited to physiological saline; With a kind of adjuvant,, but be not limited to incomplete Freund's adjuvant as incomplete Freund's adjuvant (IFA).The white powder HPV polypeptide that is loaded on phial can be stored in 4 ℃-20 ℃ and face with before being dissolved in stroke-physiological saline solution, then with the incomplete Freund's adjuvant balanced mix, be used for subcutaneous injection with shaking after instrument (Votex) shake into emulsion, but be not limited to subcutaneous injection.
The present invention selects the carrier of polypeptide as the HPV vaccine, since easy as the polypeptide source of vaccine, application safety.After albumen antigen is degraded to polypeptide in vivo, be incorporated into the HLA molecule of cell surface, give corresponding T lymphocyte, inducing cell immune response by the HLA submission then.Polypeptide only is incorporated into HLA competence exertion immune induction effect later on, and this peptide species has specificity with combining of HLA.The restricted polypeptide of HLA-A2 for example can only be incorporated into A2 and can not be incorporated into other HLA hypotype.HLA-A2 is the main hypotype of HLA, accounts for 50% of total group.The HPV polypeptide vaccine that has entered at present clinical trial all is at HLA-A2 colony basically, but also has 50% non-A2 HPV patient to can not get protection.Vaccine disclosed by the invention is not only at HLA-A2 colony, also at HLA-A1, A3, A11 and A24, thereby improved crowd's fraction of coverage of vaccine.
The present invention relates to the overlapping (Overlapes) and the combination of HPV polypeptide.Immunogenic polypeptide among the present invention or epitope are 8 to 10 amino acid longs.Aminoacid sequence in some epitope part or most of identical, but one or more amino acid difference is arranged at their aminoterminal or carboxyl terminal, show as partial sequence overlapping phenomenon.It is synthetic convenient to consider simultaneously for the ease of clinical application, and the present invention utilizes sequence overlapping phenomenon that an above epitope is enrolled a polypeptide, and it is shorter than 13 amino acid most, and is the longest no longer than 22 amino acid.
In sum, the invention provides and a kind ofly compare than prior art that specificity is stronger, fraction of coverage is higher, the better HPV polypeptide vaccine of security.
In specification sheets of the present invention, polypeptide, antigen, antigenic determinant (Antigenic Determinant) and epitope (Epitope) are considered as synonym or near synonym and use mutually.
Description of drawings
Fig. 1 induces the HPV specific cell immunoreaction for the E6 and the E7 immunogenic polypeptide of HPV16 and 18 in Balb/c mouse body, show as HPV polypeptid specificity IFN γ positive T cell and produce.
Fig. 2 induces the HPV specific cell immunoreaction for the E6 and the E7 immunogenic polypeptide of HPV16 and 18 in C57 mouse body, show as HPV polypeptid specificity IFN γ positive T cell and produce.
Fig. 3 induces the HPV specific cell immunoreaction for the E6 and the E7 immunogenic polypeptide of HPV16 and 18 in the C3H mouse body, show as HPV polypeptid specificity IFN γ positive T cell and produce.
Fig. 4 can induce generation HPV polypeptid specificity T killer cell in vivo for the E6 and the E7 immunogenic polypeptide of HPV16 and 18.
Embodiment
Embodiment 1: the prediction antigenic determinant
Access the E6 and the E7 protein sequence of HPV 16 and 18 from NIH database (www.ncbi.nlm.nih.org), with Rammensee software (Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs:1
StListing, Immunogenestics, 41,178-228,1995) analyses and prediction may set up the E6 and the E7 polypeptide libraries (Peptide Library) of HPV16 and 18 as the few peptide sequence of vaccine antigen determinant.This software is formed as amino acid from chemical structure, sequence, and the binding ability of few polypeptide and MHC is predicted in the position of fixed amino acid (Anchor amino acid), gives each the amino acid marking in the sequence.Optimal fixed amino acid is given 15 fens, and the amino acid with minimum binding ability is given 1 fen, and the amino acid that does not have binding ability is given negative the branch.This software can provide total points for each few peptide sequence automatically.Set up the peptide sequence that has 2435 9 amino acid longs with this method present inventor.In the peptide sequence of each HPV hypotype and HLA hypotype, get the polypeptide that integration is in top ten and be used for screening experiment.The HLA-A2 polypeptide is got integration and is in the first two polypeptide of ten and is used for screening experiment.Totally 240 of polypeptide that are used for screening experiment of the present invention, synthetic by Harvard Medical School.
The HPV polypeptide libraries relevant data of setting up with information biology among table 1 the present invention
| HPV hypotype E6 and E7 albumen | The HLA hypotype | The predicted polypeptide sequence number |
| HPV 16E6 158a.a. | A1 A2 A3 A11 A24 | 150 150 150 150 150 |
| HPV 16E7 98a.a. | A1 A2 A3 A11 A24 | 90 90 90 90 90 |
| HPV 18E6 158a.a. | A1 A2 A3 A11 A24 | 150 150 150 150 150 |
| HPV 18E7 105a.a. | A1 A2 A3 A11 A24 | 97 97 97 97 97 |
| Sum | 2435 |
Embodiment 2: the binding ability of using affine method test polypeptide of competitive receptor-ligand and MHC
The various binding ability of 240 polypeptide to be measured that embodiment 1 obtains and HLA is judged by the affine method of competitive receptor-ligand.Mark with fixed concentration (50 μ M)
125The restricted polypeptide to be measured of MHC (Harvard Medical School) of restricted polypeptide of the MHC of I and different concns (1-50 μ M) adds reaction system (phosphate buffered saline buffer and MHC, HLA-A2 for example), in reaction system, form two kinds of mixtures, polypeptide MHC mixture promptly to be measured and
125I labeling polypeptide mixture.Polypeptide to be measured with
125The competition of I labeling polypeptide combines with MHC's.With ultra-filtration (Ultrafiltration, Microcon 30, Amicon, Beverly, MA, USA) separated free polypeptide and polypeptide MHC mixture are measured polypeptide MHC mixture then
125The I exit dose compares the exit dose of this exit dose with the polypeptide MHC mixture (sample that does not add polypeptide to be measured) that does not have competitive inhibition, asks the concentration of polypeptide to be measured when inhibition 50% labeling polypeptide combines with MHC, i.e. IC50.Polypeptide IC50 value to be measured≤10 μ M are decided to be the positive polypeptide of binding ability.Measure HLA-A1, A2, A3, restricted polypeptide of A11 and A24 and MHC binding ability with above-mentioned same method.From more than 240 kinds of HPV polypeptide, filter out 98 kinds of polypeptide that have effective binding ability with MHC.
The E6 of table 2 HPV 16 and E7 polypeptide combine experimental result with MHC
| HPV 16 | A1 | A2 | A3 | A11 | A24 | Summation |
| Tested polypeptide number is counted % in conjunction with | 20 6 30 | 40 19 47.5 | 20 11 55 | 20 12 60 | 20 8 40 | 120 56 46.7 |
The E6 of table 3 HPV 18 and E7 polypeptide combine experimental result with MHC
| HPV 18 | A1 | A2 | A3 | A11 | A24 | Summation |
| Tested polypeptide number is counted % in conjunction with | 20 3 15 | 40 18 45 | 20 6 30 | 20 10 50 | 20 5 25 | 120 42 35 |
Embodiment 3: external immunity test
Further screen 98 HPV polypeptide selecting in conjunction with testing sieve through MHC with external immunity test.External immunity test among the present invention divided for three steps: the first step is to separate the specific peripheral blood mononuclear cell of HLA (PBMC), and second step was to cultivate altogether with HPV polypeptide and PBMC, and the 3rd step was the IFN gamma cells factor of measuring in the culture supernatant.
With FICOLL density gradient centrifugation (Boyum A:Scand J Clin Lab Invest 21 (Supp197): 77,1968) separate normal people (HLA-A1, A2, A3, A11 and A24) peripheral blood mononuclear cell (PBMC), with RPMI1640 complete culture solution (10% people AB serum RPMI1640 nutrient solution, 5ml penicillin/streptomycin, 5ml L-L-glutamic acid, 0.5ml 2 mercapto ethanol (mercaptoethanol)) adjust PBMC in 2 * 10
6/ ml.Every hole kind adds 1ml and contains the PBMC nutrient solution on the 48-orifice plate, and adding the HPV polypeptide stimulates, and peptide concentration is 10 μ g/ml.Control group only adds nutrient solution and does not add the HPV polypeptide.Cell is at 37 ℃, 5%CO
2Cultivated 21 days, 7 days is one-period.At the first thorn flyback cycle, polypeptide directly adds the PBMC moderate stimulation and cultivates.Second and the tristimulus cycle, stimulate with the antigen presenting cell that polypeptide (peptide-pulsed) is housed and to cultivate.Specific practice is as follows: at the 7th and 14 day, adding 1ml concentration in the every hole on the new 48-orifice plate was 2 * 10
6/ ml from body PBMC, incubated at room 1 hour.With PBS flush away non-adherent cell, add 10 μ g/ml polypeptide in PBS incubated at room 2 hours.Collect by stimulated cells from the culture plate in last stimulation cycle.Cell is used the resuspended every porocyte of 1ml nutrient solution after washing, and with the new culture plate of cell seeding in the antigen presenting cell that polypeptide is installed.The 2nd day adding 10 IL2 (R﹠amp of unit at each thorn flyback cycle; D Systems, Minneapolis, MN 55413, USA) in nutrient solution.Stopped cell cultures at the 21st day.
Embodiment 4 ELISA
We use ELISA to detect the HPV polypeptid specificity IFN gamma cells factor.This ELISA experimentation divides two stages: stimulation period and ELISA stage.
One. stimulation period
Collect through above-mentioned three cycle H PV polypeptide stimulated cells, the washing back adjusts to 1 * 10 with fresh medium with cell
6Cell/ml is as the effector cell of ELISA stimulation period.Settle the related HPV infection polypeptide in antigen presenting cell (from body adhesivity PBMC).Control group is not for settling the antigen presenting cell of any polypeptide and the irrelevant polypeptide of arrangement.To place the 96-orifice plate through the antigen presenting cell and the effector cell of above-mentioned processing, 37 ℃, 5%CO
2Hatched jointly 2-4 hour.Whether the collection supernatant is measured IFN γ with ELISA release can produce the immune response of HPV polypeptid specificity with the effector cell who judges the repetitious stimulation of process HPV polypeptide, and then judges whether tested polypeptide has immunogenicity.
Two .ELISA stages
(mouse-anti people IFN γ PharMingen) to 2 μ g/ml, adds antibody that 50 μ l diluted in every hole of elisa plate, is spent the night in 4 ℃ of bags to dilute capture antibody with binding soln.Wash plate to remove unconjugated capture antibody with PBS/Tween.Every hole adds 200 μ l confining liquids, and cover plate was also placed 2 hours in room temperature.Wash per three holes of with PBS/Tween and add 50 μ l same standard IFN γ, sample (above-mentioned culture supernatant) and blank, reaction is 2-4 hour under the room temperature.It is inferior to remove unconjugated IFN γ to give a baby a bath on the third day after its birth with PBS/Tween.With the biotin labeled mouse-anti people of diluted IFN gamma antibodies (PharMingen) to 1 μ g/ml.Every hole adds 50 μ l dilution back antibody, behind the cover plate in room temperature reaction 1 hour. give a baby a bath on the third day after its birth time to remove unconjugated biotin labeled mouse-anti people IFN gamma antibodies with PBS/Tween.With diluted Streptavidin-HRP to 1: 4,000.Every hole adds the Streptavidin-HRP of 50 μ l dilution.Behind the cover plate room temperature reaction half an hour.Wash 5 times to remove unconjugated Streptavidin-HRP with PBS/Tween.Every hole adds 50 μ lABTS/3%H
2O
2Room temperature reaction half an hour.
Read the OD value in every hole at 405nm with the ELISA reading machine.Calculate the concentration (pg/ml) of IFN γ according to typical curve.The judgement of positive findings: the multiple of concentration average of establishing the negative control group IFN of the concentration average γ of polypeptide experimental group IFN γ is a stimulation index, the positive result in stimulation index 〉=2.0.
So far, the present invention filters out 41 kinds immunogenic HPV polypeptide, can have the possibility as vaccine at external evoked HPV polypeptid specificity t cell immune response.
Table 4 HPV 16 and 18 E6 and the external immunization experiment result of E7 polypeptide
| HPV16&18 | A1 | A2 | A3 | A11 | A24 | Summation |
| Tested polypeptide is counted positive findings and is counted % | 9 4 44.4 | 37 16 43.2 | 17 6 35.3 | 22 8 36.4 | 13 7 53.8 | 98 41 41.8 |
Immunity test in the embodiment 5 mouse bodies
We confirm the immunogenicity of 41 polypeptide filtering out through the bioinformation prediction procedure, in conjunction with test and external immunity test at last with immunity test in the mouse body.As described in the 6th page, convenience for future products production and clinical application, also in order to reduce test sample, overlapping (Overlapes) according to polypeptid acid sequence becomes ten polypeptide with 20 left and right sides amino acid longs with 41 polypeptides in combination with 9 amino acid longs, and called after HPV polypeptide 1,2,3 respectively ... 10.With these ten immune Balb/C of polypeptide difference, C57 and C3H mouse (Medical University Of Anhui experimentation on animals center).Metapedes pad at 8-10 female mice in age in week is injected 50 μ g HPV polypeptide and adjuvant (IFB) mixed emulsion respectively, three mouse of every group of injection.Control group is injected 50 μ l PBS; Carry out booster immunization after two weeks one time, kill mouse after around the and get its spleen and separate its T lymphocyte and be used for immunoassay.Detect have there are not and have how many HPV polypeptid specificity T cells generations (HPV polypeptid specificity T cell frequency) in the mouse body with immunoenzyme connection spot technology (ELISPOT).With
51The Cr method for releasing is analyzed HPV polypeptid specificity cytotoxic T lymphocyte killing ability (CTL).
Embodiment 6 ELISPOT
Every hole adds 150 μ l confining liquids (RPMI1640 complete culture solution), hatches 2 hours with the reaction of blocking-up nonspecific binding site for 37 ℃; Settle the related HPV infection polypeptide in antigen presenting cell (homology splenocyte), hatched 1 hour for 37 ℃; Prepare the corresponding antigen presenting cells respectively with contained polypeptide in every group; The homology splenocyte of handling with irrelevant polypeptide with without polypeptide is as negative control; Shine this homology splenocyte with 3000 rad radiation doses; Resuspended homology splenocyte through above-mentioned processing is in the cell of 2 * 106/ml; Collect effector cell's (T lymphocyte) in the T-cell culture fluid, adjusting cell concn is 2 * 10
6/ ml; According to contained polypeptide number in every group with the corresponding break into portions of effector cell; Abandon confining liquid and add 100 μ l T lymphocytes and 100 μ l antigen presenting cells in the ELISPOT plate; Make positive control with 0.5 μ g/ml ConA (Sigma); 37 ℃, 5%CO
2Room temperature reaction 24 hours; From incubator, take out the ELISPOT plate and abandon cell; Wash plate 6 times with PBS/0.05%Tween 20; Every hole adds 100 μ l biotin labeled mouse-anti people IFN gamma antibodies (3ug/ml) (PharMingen), and 37 ℃ were reacted 2 hours; Finishing preceding 30 minutes preparation Avidin-Peroxidase-mixtures (APC/Vector), promptly add 1 solution A and 1 solution B in 10ml PBS/Tween 0.1% with two anti-reactions.Mix, put room temperature at least 30 minutes; After the two anti-reactions, wash plate 6 times with PBS/Tween 0.05%; Every hole adds 100 μ lAPC and in room temperature reaction 1 hour; Abandon APC and wash plate 3 times, then wash 3 times with PBS with PBS/Tween 0.05%; Every hole adds 100 μ l substrates (AEC) and room temperature reaction 4 minutes.Use the aquae destillata termination reaction; Remove the base plate of ELISPOT plate; With thieving paper culture hole is blotted, and air-dry overnight in the dark.
Numeration spot number under dissecting microscope, a spot is represented a HPV polypeptid specificity IFN γ positive T cell; With the spot number conversion is spot number (SPC/1 * 10 in per 1,000,000 T cells
6).If the spot number in per 1,000,000 splenocytes of treatment group is the twice of negative control group spot number, then the result is positive.Its result shows in the HPV polypeptide of external immunization screening, has 38 (92.7%) polypeptide can induce the generation of HPV polypeptid specificity T cell in the mouse body.
Fig. 1 shows an experiment in the immunity test in above-mentioned a series of mouse body: the disconnected neck of three one group HPV polypeptide immune and non-immunity (only injecting PBS) mouse is put to death; Take out their spleen and separate the T lymphocyte; Detect the generation of HPV polypeptid specificity IFN γ positive T cell with IFN γ ELISPOT.The T lymphocyte is basal level (Baseline) value in the value of having only nutrient solution not have to produce under the polypeptide situation in the Short-term Culture of ELISPOT, as negative control, is higher than the positive reaction of negative control value twice.The T lymphocyte of HPV polypeptide immune group produces the immune anamnestic reaction (Recall response) of significant HPV antigen-specific when running into the HPV polypeptide once more, show as the generation of IFN γ.The immune anamnestic reaction of the T lymphocyte of HPV polypeptide immune group to not producing the HPV antigen-specific under stimulating at irrelevant polypeptide.The T lymphocyte of non-immune group shows as negative immune response equally to the HPV polypeptide.HPV16 that Fig. 1 explanation is invented according to embodiment of the present invention and 18 E6 and E7 immunogenic polypeptide can be induced the HPV specific cell immunoreaction in vivo, show as the generation of HPV polypeptid specificity IFN γ positive T cell.
Embodiment 7: mouse cells in vivo toxic T lymphocyte is analyzed (CTL)
One. the priming effect cell
The spleen lymphocyte of the Balb/c mouse that separation HPV polypeptide immune is crossed, preparation individual cells suspension, the lymphocyte of interior three spleens is organized in merging; With RPMI-1640 complete culture solution washing effect cell, diluting cells concentration to 5 * 10
5/ ml; With preparing cellular control unit (not immune mouse spleen lymphocyte) with quadrat method.
Two. prepare target cell
Results P815 cell (ATCC) is with RPMI-1640 complete culture solution washing P815 cell, diluting cells concentration to 5 * 10
5Behind/the ml cell added 96 orifice plates, every hole 3ml; Adding the HPV polypeptide is 50 μ g/ml in the P815 cell to final concentration, and the P815 cell that does not add any polypeptide and irrelevant polypeptide is a control group; 37 ℃, 5%CO
2Results P815 cell after the overnight incubation is with RPMI-1640 complete culture solution washing P815 cell, centrifugal 1 * 10
7Cell (～200g, room temperature, 5 minutes) is inhaled and is removed supernatant, only stays about 0.1ml nutrient solution in pipe, and the mixing cell adds 0.2～1mCi/ml
51Cr and 20 μ l calf serums (FCS) are in above-mentioned cell; In 37 ℃, 5%CO
2Placed 60 minutes; Remove for twice unlabelled with RPMI-1640 complete culture solution washed cell
51Cr, regulating cell concn is 2 * 10
4/ ml is standby.
Three .CTL reaction assay:
Add 100 μ l effector cells and target cell respectively in round bottom 96 orifice plates, effector cell/target cell ratio is 25: 1, and 12.5: 1,6.25: 1,3.125: 1,1.56: 1, every increment originally repeated 3 holes; Contrast is established to add 100 μ l 1%Triton X-100 (maximum isotropic substance burst size) in the 100 μ l target cells of 3 holes and establish and is added 100 μ l nutrient solutions (spontaneous isotropic substance burst size), 37 ℃, 5%CO in the 100 μ l target cells of 3 holes
25 hours, centrifugal and collect every hole 40 μ l supernatant liquors and measure the sample isotropic substance burst size that the cell-specific dissolving causes that hits in γ numeration instrument; Target cell SL percentage formula: SL %=(sample isotropic substance burst size-spontaneous isotropic substance burst size/maximum isotropic substance burst size-spontaneous isotropic substance burst size) * 100.
The result who is shown in Fig. 4 has confirmed effectively killing and wounding target cell (the experimental group P815) that has the HPV polypeptide antigen, and it is very low to the lethality of the target cell (control group P815) that do not have the HPV polypeptide antigen, having confirmed that HPV polypeptide of the present invention can be induced in vivo produces HPV polypeptid specificity T killer cell (CTL), these HPV polypeptid specificities T killer cell can be discerned and have the antigenic target cell of HPV, and is killed and wounded removing.Because ctl response plays a crucial role in removing HPV cells infected and tumour cell, therefore HPV immunogenic polypeptide of the present invention can be developed and be used as prevention and therapeutic vaccine, prevention and treatment HPV infect and infect the venereal disease that causes and good, malignant tumour by HPV.
Sequence table
＜110〉Song Shuo Wang Yuan
＜120〉human papillomavirus immunogenic polypeptide, its preparation method and application
<130>
<160>33
<170>PatentIn version 3.2
<210>1
<211>17
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>1
Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro
1 5 10 15
Arg
<210>2
<211>19
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>2
Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr
1 5 10 15
Glu Leu Gln
<210>3
<211>17
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>3
Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp
1 5 10 15
Leu
<210>4
<211>14
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>4
Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys
1 5 10
<210>5
<211>14
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>5
Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr
1 5 10
<210>6
<211>21
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>6
Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu
1 5 10 15
Gln Gln Tyr Asn Lys
20
<210>7
<211>16
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>7
Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln
1 5 10 15
<210>8
<211>19
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>8
Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu
1 5 10 15
Tyr Cys Tyr
<210>9
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>9
Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu
1 5 10 15
Gln Leu Asn Asp
20
<210>10
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>10
Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His
1 5 10 15
Tyr Asn Ile Val
20
<210>11
<211>16
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>11
Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys
1 5 10 15
<210>12
<211>16
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>12
Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr
1 5 10 15
<210>13
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>13
Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr
1 5 10 15
Leu Gly Ile Val
20
<210>14
<211>19
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>14
Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val
1 5 10 15
Cys Pro Ile
<210>15
<211>17
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>15
Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys
1 5 10 15
Pro
<210>16
<211>13
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>16
Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro
1 5 10
<210>17
<211>18
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>17
Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu Cys Thr
1 5 10 15
Glu Leu
<210>18
<211>17
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>18
Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp
1 5 10 15
Ile
<210>19
<211>21
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>19
Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile
1 5 10 15
Thr Cys Val Tyr Cys
20
<210>20
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>20
Thr Cys Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu
1 5 10 15
Phe Ala Phe Lys
20
<210>21
<211>14
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>21
Glu Val Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val Tyr
1 5 10
<210>22
<211>14
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>22
Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu
1 5 10
<210>23
<211>18
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>23
Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr
1 5 10 15
Gly Leu
<210>24
<211>18
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>24
Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg
1 5 10 15
Cys Leu
<210>25
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>25
Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys
1 5 10 15
Gln Lys Pro Leu
20
<210>26
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>26
Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu
1 5 10 15
Pro Gln Asn Glu
20
<210>27
<211>21
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>27
Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu
1 5 10 15
Ile Pro Val Asp Leu
20
<210>28
<211>18
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>28
Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His
1 5 10 15
Glu Gln
<210>29
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>29
Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His
1 5 10 15
Leu Pro Ala Arg
20
<210>30
<211>15
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>30
Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro
1 5 10 15
<210>31
<211>22
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>31
Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys Cys
1 5 10 15
Glu Ala Arg Ile Glu Leu
20
<210>32
<211>20
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>32
Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe
1 5 10 15
Val Cys Pro Trp
20
<210>33
<211>19
<212>PRT
<213>Human Papillomavirus(Homo Papillomavirus)
<400>33
Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro Trp Cys Ala
1 5 10 15
Ser Gln Gln
Claims (10)
1. human papillomavirus immunogenic polypeptide, it has the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.33 in the sequence table.
2. the preparation method of the described human papillomavirus immunogenic polypeptide of claim 1: it is characterized in that this method may further comprise the steps:
(1) access human papillomavirus 16 from protein library, 18-E6 and E7 protein sequence are predicted HLA-A1 with bioinformatic analysis, A2, and A3, the restricted antigenic determinant of A11 and A24 is set up HPV16,18-E6 and E7 polypeptide libraries;
(2) filter out and HLA-A1 from this polypeptide libraries with the affine method of inhibition competition receptor-ligand, A2, A3, A11 and A24 have the polypeptide of binding ability;
(3) further filter out with external immunization and have immunogenic polypeptide;
(4) further screen with immunization in the body and confirmed that these immunogenic polypeptides induce HPV16, the ability of 18-E6 and E7 polypeptid specificity t cell immune response in vivo.
3. the described human papillomavirus immunogenic polypeptide of claim 1 is used to prepare prevention and treats the application that the human papillomavirus infects the vaccine of caused infectious diseases and neoplastic disease.
4. human papillomavirus immunogenic polypeptide according to claim 3 is used to prepare prevention and treats the application that the human papillomavirus infects the vaccine of caused infectious diseases and neoplastic disease, and it is characterized in that: described infectious diseases is human parillomarvirus infections venereal disease.
5. human papillomavirus immunogenic polypeptide according to claim 3 is used to prepare prevention and treats the application that the human papillomavirus infects the vaccine of caused infectious diseases and neoplastic disease, it is characterized in that: described neoplastic disease comprise the human papillomavirus infect due to uterine cervix, vulva, anus, penis and oropharynx proliferative lesion, innocent tumour, precancerous lesion and cancer thereof.
6. the described human papillomavirus immunogenic polypeptide of claim 1 is used to prepare the application that the detection human papillomavirus infects the reagent of caused infectious diseases and neoplastic disease.
7. the human papillomavirus prevents and therapeutic vaccine, and it is characterized in that: this vaccine consists essentially of the described human papillomavirus immunogenic polypeptide of claim 1.
8. human papillomavirus's prevention according to claim 7 and therapeutic vaccine, it is characterized in that: described vaccine also comprises adjuvant or other complementary polypeptide.
9. human papillomavirus's prevention according to claim 7 and therapeutic vaccine, it is characterized in that: described adjuvant is inorganic adjuvant, organic adjuvant, bacterium adjuvant, Nuclec acid adjuvants, or cytokine adjuvant.
10. human papillomavirus's prevention according to claim 8 and therapeutic vaccine, it is characterized in that: described complementary polypeptide is the restricted polypeptide of MHC II.
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| CN103288931A (en) * | 2012-02-24 | 2013-09-11 | 宋硕 | Immunogenic polypeptides for human papillomavirus as well as preparation method and application of same |
| CN103940993A (en) * | 2013-01-23 | 2014-07-23 | 艾托金生物医药（苏州）有限公司 | Antigen and kit for detecting anti-human-papillomavirus antibody and application of antigen |
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| CN103288931A (en) * | 2012-02-24 | 2013-09-11 | 宋硕 | Immunogenic polypeptides for human papillomavirus as well as preparation method and application of same |
| CN103288931B (en) * | 2012-02-24 | 2015-05-27 | 宋硕 | Immunogenic polypeptides for human papillomavirus as well as preparation method and application of same |
| CN103940993A (en) * | 2013-01-23 | 2014-07-23 | 艾托金生物医药（苏州）有限公司 | Antigen and kit for detecting anti-human-papillomavirus antibody and application of antigen |
| WO2014127741A1 (en) * | 2013-02-22 | 2014-08-28 | 艾托金生物医药（苏州）有限公司 | Antigen for detecting anti-human papilloma virus antibodies, test kit and applications thereof |
| CN106478784A (en) * | 2013-02-22 | 2017-03-08 | 艾托金生物医药（苏州）有限公司 | For detecting antigen and the related immune detection kit of anti-human papilloma virus (anti-HPV) antibody |
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| CN106632617B (en) * | 2013-02-22 | 2020-07-03 | 艾托金生物医药（苏州）有限公司 | Antigen for detecting anti-human papilloma virus antibody and related immunoassay kit |
| CN105753980A (en) * | 2016-03-29 | 2016-07-13 | 上海市普陀区中心医院 | HPV18 E6 monoclonal antibody and preparation method and application thereof |
| CN105753980B (en) * | 2016-03-29 | 2019-04-02 | 上海市普陀区中心医院 | A kind of HPV18 E6 monoclonal antibody and its preparation method and application |
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