Studies On Immunizing Mice With Human Papillomavirus Type 16 (Xingjiang Strain) E6 Gene Vaccines

The Study of Immunizing Mice with Human Papillomavirus Type 16 (xingjiang strain)E6 Gene Vaccines Human papillomavirus (HPV)is a dominant kind of DNA virus which causes many human tumors. Human Papillomavirus type 16 (HPV16)is thought to be a very important etiological factor causing cervical carcinoma and one of the most important highly-oncogenic ,risky HPV types. Data shows that about 50% of cervical carcinoma is related to the infections of HPV16.The early gene E6 of HPV16 encodes a transformed nonstructural virus protein. This oncoprotein is involved in malignant transformation of cervical carcinoma cells and its presence is required for the maintenance of the malignant phenotype of the tumor cells,in addition, the expression level of the E6 protein correlates closely with its transforming potential. The E6 protein is absent in the normal tissue. Therefore, if HPV16 E6 is used for construction of prophylactic and therapeutic vaccine against cervical carcinoma, it will be a kind of ideal target antigen. As the tumour specific rejective antigen, E6 can stimulate significant immunological reactions against HPV16 induced disease. This study contains: prokaryotic expression of HPV16 E6 gene isolated from some Chinese Uygur patients of cervical carcinoma in South Xinjiang, preparation and identification of HPV16 E6 rabbit/s polyclonal antibody, and immunological evaluation on mice immunized with HPV16 E6 gene vaccines. Based on standard HPV16 E6 gene sequence published in Genbank, a pair of specific primers were designed for PCR to amplify HPV16 E6 DNA from the cervical carcinoma tissues of Xinjiang Uygur women. The HPV16 E6 gene was cloned into pUCm-T cloning vector, and positive clones were analyzed with restriction enzyme digestions and further identified with sequence analysis. The sequencing results showed that the HPV16-E6 from Xinjiang Uygur women was relatively identical with the published sequence derived from the Germany strain. So the isolation of HPV16-E6 gene played the basis for the research on HPV16-E6 candidate vaccine. In order to obtain the antigen for checking antibody in mice, a constructed prokaryotic expression plasmid of pET28a-E6 presented by Dr. LI Shen-tao, was transformed into E.coli BL21 (DE3 stain) and induced by IPTG to express E6 protein. SDS-PAGE showed that the expressed E6 protein was in size of about 18KDa within prediction, and that the E6 protein expressed at high level informs insoluble solid particles---inclusionbody which contains about 90% of E6 protein under proper washing conditions. At last, the expressed E6 protein was confirmed by western bloting.The inclusionbody containing about 90% of E6 protein was further purified by SDS-PAGE, and E6 protein band on gel was cut and crushed into little particles. After being quantified, the purified E6 protein mixed with Freund,s adjuant was used to immunize Zelanian rabbits for preparing antiserum on several times. The results showed the titer of this rabbit/s polyclonal antibody was 1:20000 by ELISA,and this serum reacted specifically with HPV16E6 proteins from prokaryotic and eukaryotic expression system. To study the immune effect of mice immnunized with pCDNA3 - HPV16E6 gene vaccine, HPV16E6 genes were subcloned into a eukaryotic expression vector of pCDNA3.0. Female mice were inoculated intramuscularly with these DNA vaccines to evaluate the immune responses of DNA vaccine. Mice sera were collected to analyze their specific humoral immunity by ELISA. The results showed that HPV16E6 gene vaccine activated significantly humoral immunoresponse against the tumour specific rejective antigen HPV16 E6. These above results may help to further the research on HPV16E6 prophylactic and therapeutic vaccine against cervical carcinoma.