人乳头瘤病毒16型E7基因的原核克隆与表达

Cloning and expression of HPV16 E7 gene

目的:研究人乳头瘤病毒(Human papillomavirus HPV)16型E7在原核表达系统中的表达情况和活性,为制备HPV16型E7蛋白疫苗奠定实验基础。方法:通过聚合酶联反应(Polymerase Chain Reaction,PCR)从HPV16 DNA阳性的宫颈癌组织中扩增HPV16 E7全长基因,进一步将其克隆入原核表达载体pET32a(+),并构建重组质粒pET32a(+)/HPV16E7,经测序鉴定后转化大肠杆菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达的融合蛋白,用镍螯合亲和层析胶体(Ni-NTA Agarose)纯化,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹法(Western blot,WB)分析鉴定。结果:测序证明成功构建重组质粒pET32a/HPV16E7,IPTG诱导下HPV16E7融合蛋白在大肠杆菌中得到高效表达,重组蛋白的表达量占菌体总蛋白的16%,蛋白质印迹鉴定重组E7蛋白与表达载体的标签蛋白形成相对分子质量约30×103的可溶性融合蛋白。结论:重组质粒pET32a/HPV16E7在大肠杆菌BL21中高效表达目的蛋白。

Objective：To study the expression and activity of the gene E7 protein of human papillomavirus in the expression system of the prokaryon and built an experimental foundation for preparing the E7 recombinant protein. Methods：HPV16E7 gene amplified by PCR was cloned into the expression vector pET32a to form recombinant plasmid pET32a/HPV 16ET,then transformed into E. coli BL21. The pET32a/HPV16 E7 fusion protein was induced by IPTG and purified with Ni-NTA gel chromatography column. The protein was verified with SDS-PAGE and Western blot. Results：The recombinan plasmid was identified and confirmed with double enzymes digestion and sequencing. It was suggested that the recombinant E7 protein was expressed with SDS-PAGE and western blot, and a molecular weight was about 30 × 10^3. The fusion protein was about 16% of the total bacterial protein. Conclusions：Highly expressed E7 protein of HPV16 is obtained.