中国医学科学院学报2011，Vol.33Issue(1)：98-101,4.DOI:10.3881/j.issn.1000-503X.2011.01.022
丙型肝炎病毒蛋白酶抑制剂高通量筛选模型的建立及应用
Establishment and Application of High Throughput Screening Model for Hepatitis C Virus NS3 - 4A Protease Inhibitors in vitro
摘要
Abstract
Objective To establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro. Methods HCV recombinant plasmid pMAL-c2/NS3-4A was transformed into the E. coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated. Results High throughput screening model for HCV NS3-4A protease inhibitors was established,and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0. 80, and coefficient of variation (CV) was 1.91％. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L. Conclusion The assay model using FRET method for HCV NS3-4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3-4A protease inhibitors.关键词
丙型肝炎病毒/丝氨酸蛋白水解酶/高通量筛选模型/荧光共振能量转移分类
医药卫生引用本文复制引用
李健蕊,武燕彬,司书毅,陈鸿珊,蒋建东,彭宗根..丙型肝炎病毒蛋白酶抑制剂高通量筛选模型的建立及应用 [J].中国医学科学院学报,2011,33(1):98-101,4.基金项目
科技部国际合作项目（2006DFA31450）、国家新药创制重大专项（2009ZX09302-004）和中央级公益性科研院所基本科研业务专项 （）
