The Study Of DNA Immunization With HPV16 E7 Gene Isolated From Xinjiang Uygur Women Cervical Carcinoma

Evidence assigns significance to the high-risk HPV infection in the occurrence of the cervical cancers in human. It is clear that the E7 derived from the high-risk HPV16 encodes the predominant transforming protein, which is constitutively expressed in the cervical carcinoma tissues. Therefore, the E7 gene is regarded as an important candidate target for prophylactive and therapeutic HPV vaccine development.In this study, HPV16-E7 gene was isolated from cervical tissues of Xinjiang Uygur women and cloned into eukaryotic expression vector as the prototypic DNA vaccine, pCDNA3-E7,which was used to immunize animals. Enhancement of the immune effects on these animals specific to E7 was observed with two molecular adjuvants, IL-2 gene and CpG containing oligonucleotide,which were co-immunized with pCDNA3-E7. The fusion protein of GST-E7 was expressed in E. coli and purified used for ELISA detection.To isolate HPV16-E7 gene from local cervical carcinoma specimen, a pair of PCR primers was designed according to the published HPV16-E7 sequence. After PCR of DNA isolated from the cervical carcinoma tissues of Xinjiang Uygur women, the HPV16-E7 gene was cloned into cloning vector, pUCm-T. Positive clones were analysed with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the HPV16-E7 from Xinjiang Uygur women was identical with the published sequence derived from the Germany strain (GI:9627100).A prokaryotic expression construct, GST-2T-E7, obtained from Dr. Werner was transformed into E.coli BL21 and induced by IPTG to express GST-E7 fusion protein. The expressed GST-E7 fusion protein was purified from bacterial lysates by affinity chromatography using glutathione-agarose resin and used in ELISA analysis as the antigen.For construction of DNA vaccine, pCDNA3-E7, the cloned HPV16-E7 gene was subcloned into an eukaryotic expression vector, pCDNA3. To evaluate the immune responses of this construct, animals were inoculated intramuscularly by the pCDNA3-E7 and sera were collected to analyzed their specific antibodies against E7 using ELISA assays. To determine the enhancement of immune response for this DNA vaccine, eukaryotic expression vector contains the IL-2 gene and oligonucleotide contains the CpG motif were used to co-inoculated into animals with the pCDNA3-E7.The ELISA results showed that the anti-HPV16E7 antibody was elicited specifically against E7 antigen after immunization of pCDNA3-E7, whereas the pCDNA3-IL-2 vector and CpG motif have marked effects on level of antibodies induced by the pCDNA3-E7 DNA vaccine in these tested animals. These results suggest that constructed pCDNA3-E7 can elicit specific anti-E7 antibody in animal and the observed immune responses can be augmented by co-inoculation with IL-2 gene and CpG oligonucleotide. These preliminary results could lead to further investigating the feasibility for HPV16 vaccine development.