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目的 构建丙型肝炎病毒 (HCV)NS3基因的原核细胞表达载体 ,实现在大肠埃希菌中的可诱导性表达。方法 应用聚合酶链反应 (PCR)技术 ,以美国HCV H株全长cDNA质粒为模板 ,扩增获得NS3基因片段 ,克隆到原核表达载体pET 30C(+)中 ,构建原核表达载体pET NS3,转化BL2 1(DE3)宿主菌 ,以IPTG诱导 ,获得NS3蛋白的可诱导性表达 ,以HCVNS3的单链可变区抗体 (ScFv)证实表达的NS3蛋白的特异性。结果 以HCVNS3基因序列特异性引物 ,PCR扩增获得 1893bp的NS3DNA片段 ,插入pET 30C(+)表达载体。转化BL 2 1(DE3)受体菌 ,经培养、IPTG诱导 ,获得了重组HCVNS3蛋白的表达 ,以HCVNS3的ScFv证实了表达的重组蛋白HCVNS3的特异性。结论 以大肠埃希菌表达了HCVNS3的重组蛋白质
Objective To construct prokaryotic expression vector of hepatitis C virus (NS3) gene and achieve its inducible expression in Escherichia coli. Methods The full length cDNA of HCV H strain was used as a template to amplify the NS3 gene fragment by polymerase chain reaction (PCR) technology and cloned into the prokaryotic expression vector pET 30C (+). The prokaryotic expression vector pET NS3 was constructed and transformed BL21 (DE3) host strain induced by IPTG to obtain inducible expression of NS3 protein. The specificity of NS3 protein expressed was confirmed by single chain variable region antibody (ScFv) of HCV NS3. Results The 1893bp NS3 DNA fragment was amplified by PCR using the sequence-specific primers of HCV NS3 gene and inserted into pET 30C (+) expression vector. The recombinant BL21 (DE3) was transformed into E.coli BL21 (DE3), and induced by IPTG. The expression of recombinant HCVNS3 protein was obtained. The specificity of recombinant HCVNS3 was confirmed by ScFv of HCVNS3. Conclusion The recombinant protein of HCV NS3 was expressed in Escherichia coli