酶联免疫吸附试验检测HCV NS3-4A蛋白酶活性

Detection of Proteolytic Activity of Hepatitis C Virus NS3-4A Protease Using Enzymelinked Immunosorbent Assay

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。

To establish an ELISA method for detecting proteolytic activity of HCV serine protease for screening inhibitors against HCV.Methods HCV recombinant plasmid pMAL-c2/NS3-4A was transformed into the E.coli strain K 12TB 1.Maltose-binding-protein(MBP)-NS3/NS3-4A fusion protein expression were induced by adding isopropyl-β-D-thiogalacto-pyranoside(IPTG)and purified by affinity chromatography.The immunological activities of the fusion protein were analyzed by Western blot.A peptide substrate was used to analyze the biological activity of the fusion protein.The hydrolyzed product was treated with sodium iodacetate and labeled with digoxigenin,then adding immunological anti-digoxigenin-alkaline phosphatase conjugate to detect the protease activity by colorimeteric reaction.Results The purified MBP-NS3 and MBP-NS3-4A proteases were identified as 112 000 u and 116 000 u protein by Western blot.HCV NS3/NS3-4A protease showed substrate cleavage activities by ELISA.Coefficients of variation(CV)of ELISA in a lot and among the lots were 4.16% and 7.52% respectively,the P/N was 3.63 under the best experimental conditions determined by L 9(3 4)factorial design.The method confirmed that 8.36 μmol/L 1,4-naphthoquinone had 50% inhibitive activity on HCV serine protease.Conclusions We have established a simple and rapid ELISA method with stable repeatability for detecting proteolytic activities of HCV NS3-4A protease,which might be used for screening and studying of specific inhibitors of HCV serine protease NS3-4A.