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目的研究丙型肝炎病毒(HCV)聚合酶底物及pH值最佳范围。方法构建HCV聚合酶基因的重组原核表达质粒后,测序及读码框架分析结果表明得到相关HCVNS5bRNA聚合酶全基因序列,进一步利用Ni2+金属离子螯和亲和层析将其纯化。结果研究获得高效表达聚合酶蛋白的最适条件,利用DNA-BAND培养96孔板,建立生物素标记检测HCVNS5b聚合酶活性的模型。同时研究细胞外丙型肝炎病毒聚合酶复制模型使用的HCV聚合酶作用底物及pH值的最佳条件。结论HCV聚合酶的高效表达和有效纯化,以及HCV聚合酶的底物及pH值对聚合酶的最佳条件的确定为建立细胞外丙型肝炎病毒聚合酶复制模型及筛选药物奠定基础。
Objective To study the optimum range of substrate and pH value of hepatitis C virus (HCV) polymerase. Methods After construction of the recombinant prokaryotic expression plasmid of HCV polymerase gene, sequencing and reading frame analysis showed that the complete HCVNS5b RNA polymerase gene sequence was obtained and purified by Ni2 + chelating and affinity chromatography. Results The optimum conditions for efficient expression of polymerase protein were obtained. The 96-well plates were incubated with DNA-BAND to establish a biotin-labeled model for detecting the activity of HCV NS5b polymerase. At the same time, the optimal conditions for the substrate and pH value of HCV polymerase used in the extracellular replication model of hepatitis C virus polymerase were also studied. Conclusion The efficient expression and purification of HCV polymerase, as well as the determination of substrate and pH value of HCV polymerase for the optimal conditions of the polymerase, lay the foundation for the establishment of a replication model of extracellular hepatitis C virus polymerase and screening of drugs.