丙型肝炎病毒非结构蛋白NS5A反式激活基因4剪切体Ns5atp4a基因原核表达载体的构建和表达

Construction of the prokaryotic expression vector of NS5ATP4A gene and its expression

目的构建丙型肝炎病毒非结构蛋白NS5A反式激活基因4剪切体(NS5ATP4A)的原核表达载体并进行表达。方法用PCR扩增NS5ATP4A基因,克隆入pET32a(+)质粒,构建pET32a(+)-NS5ATP4A表达重组体,转化DH5α和BL21菌,经IPTG诱导。SDS-PAGE分析,Western Blot验证蛋白带。结果成功构建大肠杆菌原核表达载体。经IPTG诱导,得到了目的蛋白分子量为35kD,用Western Blot证实其具有良好的抗原性。结论构建原核表达载体pET32a(+)-NS5ATP4A,为抗原的制备及临床检测奠定了基础。

Objective To construct prokaryotic expression vector of NS5ATP4A gene and express it in E.coli.Methods The NS5ATP4A gene was amplified by PCR and cloned in to plasmid pET32a（＋） to construct recombinant prokaryotic expression vector pET32a（＋）-NS5ATP4A.After transfection into the E.coli and induction with IPTG,recombinant target protein was analyzed by SDS-PAGE and approved by Western Blot.Results The recombinant prokaryotic expression vector was constructed successfully.The antigenicity of 35KD target protein,induced by IPTG was proved by Western Blot.Conclusion The construction of prokaryotic expression vector pET32a（＋）-NS5ATP4A provides the foundation for the preparation of antibody in clinical test.