丙型肝炎病毒RNA依赖的RNA聚合酶在大肠杆菌中可溶性表达条件的优化  

Optimization on Soluble Expression of Hepatitis C Virus RNA-Dependent RNA Polymerase in E.coli

作　　者：李琳[1] 潘英[2] 王正茂[1] 傅雅丽[1] 李越希[1,2] 

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》2010年第3期207-211,共5页Pharmaceutical Biotechnology

基　　金：江苏省自然科学基金;项目编号:BK2007014

摘　　要：为了提高丙型肝炎病毒RNA依赖的RNA聚合酶蛋白(HCV-RdRp)在大肠杆菌中的可溶性表达,对影响蛋白可溶性表达水平的多种因素进行了优化。探讨了诱导时机、诱导温度及时间、诱导剂IPTG浓度等对重组蛋白可溶性表达的影响,筛选出了提高可溶表达该蛋白的最适条件。结果表明,在菌体密度OD600约为1.6时,加入终浓度为0.20 mmol/L的诱导剂IPTG,25℃下诱导表达16 h,可以获得最多的可溶性表达蛋白,获得了纯化较好并有特异性的HCV-RdRp重组蛋白,为建立其体外活性测定方法及抑制剂的筛选奠定了基础。In order to improve the soluble expression of recombinant Hepatitis C virus RNA-dependent RNA polymerase （RdRp） in E. coli, several factors influencing RdRp expression level and solubility were optimized by using single factor tests. The expressed RdRp was detected by SDS PAGE. The results showed that amplification cultural time, IPTG concentration, induction temperature and induction time have significant effects on solubility of the expressed RdRp. The best condition for express ing soluble RdRp was obtained, when OD600 of the culture medium reached about 1. 6, and the engineered E. coli was induced with 0.20 mmol/L IPTG at 25 ℃ for 16 h. This study has established the foundation for assaying RdRp enzymatic activity in vitro and screening the anti HCV drugs targeting RdRp.

关 键 词：丙型肝炎病毒 RNA依赖的RNA聚合酶 可溶性表达 优化 

分 类 号：R512.63[医药卫生—内科学]