【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。更多还原

【Abstract】 Objective:To construct a prokaryotic expression plasmid containing HCVF 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 1a subtype),cloned into pET32a(+)vector,and then transformed into E.coli JM109. After identified by restriction diges- tion 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 35.4kDprotein band was found bySDS- 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 helpful for further research.更多还原