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.%目的 应用荧光共振能量转移(FRET)法检测丙型肝炎病毒(HCV)丝氨酸蛋白水解酶(NS3 - 4A)活性,建立HCV蛋白酶抑制剂筛选模型.方法 将质粒pMAL-c2/NS3 - 4A转化到大肠杆菌K12TB1中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,亲和层析柱纯化得到麦芽糖结合的NS3 - 4A融合蛋白,用FRET法测定蛋白水解酶活性,建立特异性蛋白酶抑制剂筛选模型,优化反应体系,评价模型的可靠性.结果 建立了HCV蛋白酶抑制剂高通量筛选模型,经优化确定了酶及底物用量.在模型可靠性评价中,Z因子高达0.80,变异系数为1.91%.蛋白酶的Km值为4.74 μmol/L,测得已知HCV蛋白酶抑制剂BILN 2061的Ki值为0.30 nmol/L.结论 建立的HCV丝氨酸蛋白酶抑制剂FRET法筛选模型稳定可靠,适用于高通量筛选.
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