Cloning, Expression And Identification Of Human Papillomavirus Type-16 E2 Gene

Cervical cancer is one of the leading causes of cancer-related death in women.Evidences from Human Papillomavirus indicated that more than 90% cervical cancer development was a complex process associated with high-risk HPV infection.HPV type 16 (HPV-16) was related to it predominate.High-risk HPV infection of the cervical epithelium occurs in two forms.In acute infections,a complete copy of the HPV viral DNA is present as an episome within the host cell,and the virus is capable of completing its life cycle,producing new,infectious viral particles.In a small minority of HPV-infected patients,the circular viral genome is integrated into the host-cell DNA, producing high-grade dysplasia,which can progress to invasive carcinoma.Among these genes, HPV16E2 and HPV16E6 gene play important roles in cervical cancer regulation.The correlation among E2 gene,E6 gene and cancer cell division,as well as the E2 gene function are still suspensive.In the current study,to discuss whether there is any effect produced by E2 on cervical cancer development, E2 gene was expressed in E.coli.and purified by affinity chromatography.then anti-HPV16E2 antibody was prepared.Meanwhile,the E2 recombinant plasmid was transfected into SiHa cells,then the effects on the HPV16E6 mRNA expression and the SiHa cell growth were analysed.In this study,The SDS-PAGE results suggested that in Escherichia coli BL21, the E2 fusion protein was expressed and purified. The active immunization results showed that antibody against E2 was specific.The pCDNA3/HPV16E2 group had significant lower HPV16 E6 mRNA content and cell growth number than control group detected by Semi-Quantitative RT-PCR and cell counting.Therefore,HPV16E2,binding the early gene promoter and regulating HPVE6 gene expression, can restrain cell growth and division, which will provide the cue for cervical cancer therapy.The research comprises as follows:First, the construction of expression vector of HPV16E2 gene from HPV.To construct the prokaryotic and eukaryotic expression vector and prepare the antiserum of HPV16E2 gene. The sequence of HPV16E2 gene was amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of cervical cancer cells and insert into pMAL vector and pCDNA3 vector.Secondly,the HPV16E2 prokaryotic expression and preparation of HPV16E2 gene antiserum.To further study the function of HPV16E2 gene,HPV16E2 were cloned into pCDNA3 to form pCDNA3/HPV16E2 DNA vaccines,and then vaccinate mice by DNA delivery way.Fusion protein MBP/E2 were expressed in E.coli.BL21,into which prokaryotic expression plasmids pMAL/HPV16E2 was introduced with IPTG induction. The expressed fusion proteins were purified by MBP affinity chromatography.ELISA and Western blotting showed that antibody obtained from the serum of mice by injecting recombinant plasmid pCDNA3/HPV16E2 were strong specific.The recombinant plasmid pCDNA3/HPV16E2 and anti-HPV16E2 antibody in the current study would provide a powerful tool for function research of HPV gene in cell level;Finally,pCDNA3/HPV16E2 effects on SiHa cells growth number in detection.pcDNA3/HPV16E2 was transfected into SiHa cells and E2 protein was expressed.The HPV16E6 mRNA expression and SiHa cell growth were detected.RT-PCR and Western blotting result respectively showed that in SiHa cells E2 mRNA and protein were expressed.In HPV16E2 gene transfection test, the pCDNA3/HPV16E2 group had significant lower HPV16E6 mRNA level and cell growth number than control group(P<0.05).