丙型肝炎病毒C端截短21个氨基酸NS5B蛋白的克隆、表达及鉴定  

Cloning,Expression and Identification of HCV NS5B with C-Terminal 21 aa Truncated

作　　者：杨华凤[1] 潘明洁[2] 吕敏[1] 李越希[2] 

机构地区：[1]南京医科大学公共卫生学院流行病与卫生统计学系,江苏南京210029 [2]南京军区军事医学研究所,江苏南京210002

出　　处：《药物生物技术》2008年第2期86-89,共4页Pharmaceutical Biotechnology

基　　金：江苏省自然科学基金;项目编号:BK2007014

摘　　要：构建HCV C端截短21个氨基酸的NS5B(HCV NS5B-C21)的重组原核表达质粒,并获得HCVNS5B-C21蛋白,为以其为靶位的抗HCV药物筛选等创造条件。利用PCR技术扩增HCVNS5B-C21基因,BamHⅠ和XhoⅠ双酶酶切后连接到经同样酶酶切的原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3)菌株,获得阳性重组质粒pET-28a(+)-NS5B-C21,IPTG诱导表达,亲和层析法纯化目的蛋白表达产物经SDS-PAGE进行鉴定。成功构建了HCV NS5B蛋白表达载体pET-28a(+)-NS5B-C21,经诱导明显表达出6His-NS5B-C21,截短形式的6His-NS5B-C21表达产物的可溶性明显增加。HCV NS5B-C21蛋白在体外得到了有效表达,表达的蛋白主要存在于上清液中,纯化获得了NS5B-C21蛋白,为抗HCV药物筛选等奠定基础。To construct the engineering E, coli, expressing HCV NS5B protein with C-terminal 21 aa truncated and to acquire the protein for screening the anti-HCV drugs targeting the NSSB, The coding region of HCV NS5B-C21 gene was amplified by PCR and was digested by BamH I and Xho I , the gene fragment was cloned into plasmid pET 28a（＋） and the recombinant plasmid was transformed into E, coli BL21 （DE3）. The positive recombinant plasmid was obtained by identification of PCR amplification, and it was named pET-28a（＋）-NSSB-C21. The engineering E. coll. harboring the recombinant pasmid was induced by IPTG and analysed by SDS-PAGE, NS5B-C21 protein was efficiently expressed, and the solubility of expressed NSSB-C21 protein was increased obviously. The expressed NSSB-C21 protein was purified with Ni Affinity column, and highly pure NS5B-C21 protein was obtained successfully, which established the foundation for screening the anti HCV drugs targeting the NSSB.

关 键 词：丙型肝炎病毒 非结构区5B蛋白 RNA依赖的RNA聚合酶 克隆 表达 

分 类 号：Q7[生物学—分子生物学]