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目的 建立一种检测丙型肝炎病毒 (HCV) NS3- 4A丝氨酸蛋白酶的酶联免疫吸附试验 (EL ISA)方法 ,用于筛选 HCV蛋白酶抑制剂。方法 将质粒 p MAL- c2 / NS3- 4A转化到大肠杆菌 K1 2 TB1 中 ,经 IPTG诱导表达 ,亲和层析柱纯化后得到麦芽糖结合的 NS3/ NS3- 4A融合蛋白 ,用 Western blot分析其免疫学活性 ,并以酶联免疫吸附试验检测其生物学活性。结果 SDS- PAGE电泳分析并用考马斯亮蓝染色 ,显示相对分子质量 112 0 0 0和116 0 0 0处有麦芽糖结合的 NS3及 NS3- 4A融合蛋白带出现 ;Western blot分析表明 ,表达的产物可与抗 NS3蛋白的单克隆抗体发生阳性反应。EL ISA证实 HCV NS3- 4A蛋白酶能使特异底物裂解。正交实验获得 EL ISA的最佳实验条件 ,其 P/ N值为 3.6 3,批内和批间变异系数 (CV)分别为 4.16 %和 7.5 2 %。此方法测定 1,4萘醌对 HCV蛋白酶活性 5 0 %抑制浓度 (IC5 0 )为 8.36 μm ol/ L。结论 建立的 EL ISA方法 ,用于测定 HCV蛋白酶活性 ,具有简便、快速、重复性好等特点 ,为以 HCV NS3- 4A蛋白酶为靶酶的抑制剂筛选及抗 HCV药物的研究奠定了基础。
Objective To establish an enzyme-linked immunosorbent assay (ELISA) for the detection of HCV NS3-4A serine protease for the screening of HCV protease inhibitors. Methods Plasmid p MAL-c2 / NS3-4A was transformed into E. coli K1 2 TB1 and induced by IPTG. Maltose-conjugated NS3 / NS3-4A fusion protein was obtained after purified by affinity chromatography. Learning activity, and enzyme-linked immunosorbent assay to detect its biological activity. The results of SDS-PAGE electrophoresis analysis and staining with Coomassie brilliant blue showed that maltose-binding NS3 and NS3-4A fusion protein bands appeared at the molecular weight of 112 000 and 116 0 0 0; Western blot analysis showed that the expressed product could be used with Anti-NS3 protein monoclonal antibody positive reaction. EL ISA confirms that HCV NS3-4A protease cleaves specific substrates. The optimum experimental conditions of EL ISA obtained by orthogonal experiment were P / N of 3.6 3 and CV of 4.16% and 7.5 2% respectively. The method determined the inhibitory concentration (IC50) of 1,4-naphthoquinone to HCV protease activity of 8.36 μmol / L. Conclusion The EL ISA method was established for the determination of HCV protease activity. It is simple, rapid and reproducible. It lays the foundation for the screening of inhibitors targeting HCV NS3-4A protease and the anti-HCV drugs.