丙型肝炎病毒ns3区全长基因在大肠杆菌中的高效表达及纯化  

Highly Efficent Expression and Purification of HCV ns3 Gene in E.coli

作　　者：杨敬[1] 薛小平[1] 雷迎峰[1] 尹文[1] 吕欣[1] 徐志凯[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《生物技术通讯》2005年第1期13-15,共3页Letters in Biotechnology

摘　　要：利用PCR技术扩增HCVns3基因,经BamHⅠ和HindⅢ双酶切后与原核表达质粒pProEX-HTb连接,转化感受态细胞E.coliDH5α,酶切鉴定得阳性重组质粒pProEX-HTb-ns3并测序;pProEX-HTb-ns3转化宿主细胞获得工程菌,用IPTG诱导,获得NS3蛋白的高效表达,薄层扫描显示其占菌体总蛋白的35%;目的蛋白在变性条件下经Ni2+-NTA凝胶亲和层析纯化,透析并浓缩后用丙型肝炎患者阳性血清做为一抗行Western-Blot证实特异性和抗原性。结果成功表明,诱导表达产物主要以包涵体形式存在;6His-NTA纯化后获得目的蛋白,Western-blot结果显示纯化蛋白具有良好的抗原性。HCVNS3蛋白的高效表达及纯化,为利用NS3蛋白作为诊断抗原及制备单克隆抗体奠定了基础。Construct the recombinant plasmid expressioning HCV ns3 gene and acquire and purify the NS3 protein for diagnostic antigen and to prepare the monoclonal antibody. The HCV ns3 gene was amplified by PCR and was digested by HindⅢ+BamHⅠ. This fragment was cloned into pProEX-HTb by T4 ligase and transformed E.coli DH5α. The positive recombinant plasmid named pProEX-HTb-ns3 was obtained. The NS3 protein was expressed distinctly after inducing by IPTG. Thus the protein was purified on Ni2+-NTA column. Western-Blot performed with the positive serum from HCV patient as the first antibody identify the antigen activity of the purification NS3 protein. The recombinant plasmid pProEX- HTb-ns3 was constructed successfully; the production mainly was inclusion and purified on Ni2+-NTA column; the purification protein keep the antigen activity of NS3 protein. The highly efficient expression and purification of HCV NS3 protein lay a foundation of diagnostic antigen.

关 键 词：丙型肝炎病毒 非结构区 原核表达 纯化 抗原性 ns3基因 

分 类 号：R512.63[医药卫生—内科学] R373.21[医药卫生—临床医学]