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目的 以聚合酶链反应 (PCR)突变方法诱导丙型肝炎病毒 (HCV)蛋白酶活性位点ser116 5的突变 ,获得全长非结构基因 3(NS3) / 4a的表达与纯化。方法 分别以NS3N端正向引物与诱变反向引物 ,诱变正向引物与NS4aC端反向引物获得 2个PCR产物 ,产物纯化后在新的PCR反应体系中加入以上 2个PCR产物与NS3N端正向引物、NS4aC端反向引物。再次PCR扩增突变模板 ,分别与野生型模板重组入表达载体pET2 6 Ub ,转化大肠杆菌BL2 1(DE3)pCG1,诱导表达后经菌体裂解、纯清化、硫酸铵沉淀、DEAE Sepharose、NTA纯化 ,Westernblot分析表达蛋白的特异性及PCR诱导突变使HCV蛋白酶活性位点失活的作用。结果 获得诱导突变的模板 ,Westernblot证实该突变可完全阻断对NS3丝氨酸蛋白酶与NS3螺旋酶间的切割 ,部分阻断了螺旋酶与NS4a间的切割。纯化后的HCVNS3/ 4a蛋白在SDS PAGE胶上显示为双带。结论 PCR突变方法简便、有效 ,获得丝氨酸蛋白酶失活的NS3蛋白表达 ,NS3蛋白与NS4a蛋白以复合物形式存在
Objective To induce the mutation of ser116 5, a protease active site of hepatitis C virus (HCV), and obtain the expression and purification of the full length nonstructural gene 3 (NS3) / 4a by polymerase chain reaction (PCR) mutation. Methods Two PCR products were obtained by using NS3N forward primer, mutagenesis reverse primer, mutagenesis forward primer and NS4aC reverse primer respectively. After purification, the above two PCR products were added to the new PCR reaction system and NS3N positive Reverse primer to NS4aC. The recombinant plasmid was recombined with the wild-type template into the expression vector pET2 6 Ub and then transformed into E.coli BL21 (DE3) pCG1. The recombinant plasmid was induced to lyse, purified, ammonium sulfate precipitation, DEAE Sepharose, NTA Purification, Western blot analysis of the expression of protein specificity and PCR-induced mutation of HCV protease active site inactivation role. Western Blotting confirmed that the mutation completely blocked the cleavage between NS3 serine protease and NS3 helicase and partially blocked the cleavage between helicase and NS4a. The purified HCV NS3 / 4a protein appears as a double band on SDS PAGE gels. Conclusion The method of PCR mutation is simple and effective, and the NS3 protein inactivated by serine protease is obtained. The NS3 protein and NS4a protein exist as complex