丙型肝炎病毒F蛋白原核表达载体的构建、表达和纯化  

Prokaryotic expression,identification and purification of HCV F protein

作　　者：高亚丽[1] 刘娇[1] 刘湘[1] 汤华[1] 陈沂[1] 蔡雪飞[1] 唐霓[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室重庆医科大学病毒性肝炎研究所,重庆400016

出　　处：《重庆医科大学学报》2009年第4期385-388,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(30400374;30600405)。

摘　　要：目的:构建丙型肝炎病毒(Hepatitis Cvirus,HCV)F蛋白的原核表达载体,表达pET32a(+)-HCVF融合蛋白。方法:根据丙型肝炎病毒F基因的全序列设计引物,以HCV1a型cDNA质粒H/FL为模板,通过PCR方法扩增得到F基因的编码区序列,将其定向克隆于含有6×Histag的原核表达载体pET32a(+),转化大肠杆菌JM109,经菌落PCR筛选,双酶切和测序鉴定阳性克隆后,转化大肠杆菌BL21,采用IPTG诱导表达融合蛋白,表达产物经SDS-PAGE电泳分析和Western-blot检测鉴定,并用Ni-NTA亲和层析柱纯化。结果:F基因以正确的方式插入到pET32a(+)载体中,重组质粒转化大肠杆菌BL21后经IPTG诱导表达,出现了与预期分子量相符的蛋白条带,该蛋白通过Westernblot检测具有6×Histag蛋白的免疫学活性。结论:成功构建了丙型肝炎病毒F蛋白的原核表达载体pET32a(+)-HCVF并表达和纯化出重组融合蛋白,为进一步研究F蛋白的生物学功能奠定了基础。Objective： To construct a prokaryotic expression plasmid containing HCV F gene, and purify the recombinant fusion protein in E.coli system. Methods： F gene of Hepatitis C virus was amplified by PCR method from plasmid H/FL （containing full length cDNA sequence of HCV la subtype ）, cloned into pET32a （＋） vector, and then transformed into E.coli JMI09. After identified by restriction digestion and DNA sequencing, recombinant plasmid was transformed into E.coli BL21 and induced with IPTG. The fusion protein trxA-F was further confirmed by Western blot analysis and purified by affinity chromatography method. Results： Restriction digestion and PCR screening showed that HCV F gene was cloned into pET32a（ ＋ ） successfully. After BL21 was transformed with recombinant vector pET32a （＋）-HCVF and induced with 0.5mmol/L IPTG, a 35.4kD protein band was found by SDS-PAGE. And this recombinant protein showed a highly specific and strong reaction with anti-His monoclonal antibody by Western blot analysis. Conclusion： The prokaryotic expression plasmid pET32a（＋）-HCVF was successfully constructed, which will be heloful for further research.

关 键 词：丙型肝炎病毒 F蛋白 原核表达 

分 类 号：R512.6[医药卫生—内科学]