Expression And Purification Of HPV16L1/E7 Fusion Protein In Prokaryotic System And The Study Of Its Immunogenicity

Objective:Infection with human papillomaviruses (HPV) is highly associated with the development of cervical cancer.This disease is the second most frequent malignant female cancer worldwide, claiming more than 200,000 lives every year. Most of the cases occur in countries of the Third World where screening programs for early detection of cervical cancer do not exist. The prototype of high risk human papillomaviruses is HPV 16, the transforming potential of these viruses is best studied in the case of cervical cancer. HPV infections related to cervical cancer are mostly transmitted through sexual intercourse; hence effective immunization must take place in teenagers at an age before they become sexually active. Due to the long time lapse between infection and the onset of the tumor, it will take many years before the reduction of cancer incidence becomes visible. We are focusing on the development of vaccines which would be useful in already infected individuals and thus would yield an earlier benefit ("therapeutic vaccine"). The most promising target antigens for an attack of immune cells are the viral oncoproteins proteins E6 and E7 which are expressed in every tumor cell.Recently,someone generated chimeric virus-like particles (CVLP) of HPV 16 which assemble from 360 copies of a fusion protein between the major structural protein L1 and the 55 N-terminal amino acids of E7. CVLPs proved to highly induce the neutralizing antibodies and E7-specific cytotoxic T cells,so it have both prophylactic and therapeutic effection.But the chimeric protein expresses in prokaryotic system can not assemble to chimeric virus-like particles(cVLP) instead of pentamer.The immunologic competence of the pentamer is familiar with VLP. It is our major goal to develop optimized methods for the development of a kind of vaccine have the quality of low-cost and therapy and prophylaxis. Method: we can construct a new express vector pQE L1â–³C34E7N60 in advantage of plasmid pMD L1 â–³C34E7N60 ,and the recombinant construct was confirmed by enzyme.Then the plasmid was transformed into bacteria M15.The L1â–³C34E7N60 fusion protein have six his expression tag was expressed after induction.The protein was verified by Western blot analysis against the L1 and E7 antibodis.The fusion protein is insolvable,so we can get the inclusion body,we can make it purer by low concentration urea. Then the purification was performed by the way of ion exchange and affinity chromatography.After renaturation and concintration we can obtain the solvable target-protein.The prophylactic and therapeutic immunologic competence of the L1â–³C34E7N60 fusion protein was testified by prevention and therapeuticexperiment in vivo.In the same time, we can observe the effect of adjuvant and the relationship between the dosage of protein and the ratio of tumor occurrence.At last,the antibody titre aginst L1 and E7 in the mice in prevention experiment be measured by ELISA. Result: This study constructed the recombinant plasmid pQE L1â–³C34E7N60 and expressed the fusion protein in prokaryotic expression system successfully.The protein expressed as insolvable form and the molecular is 61 KD.The target protein was obtained after ion exchange and affinity chromagraph.The purified protein turned to solvable after renaturation and concintration.The purification was assessed at 96% using Gelpro software.We can yield almost 5 mg targeting protein in 1 liter bacteria.The result of protection against tumor challenge expriment demonstrated that all the na?ve mice developed palpable tumors.By contrast,about half of all in treat group remains tumor free.The analysis of the result have statistical significance. And there have no difference between the L1/E7 and L1/E7 plus adjuvant. In the therapueutic experiment,the occurrence of tumor have logistic association with the change of the dosage of protein.In addition,10 ug group have perfectly therapeutic effect.The high titres of L1 and E7 antibody can be detected by ELISA. Conclusions:In brief,Our results show that HPV16 L1/E7 fusion protein is high expressed in E.coil and the target protein can be purified by ion exchange and affinity...