丙型肝炎病毒聚合酶底物及pH值最佳范围研究

Study on optimal concentration of poly A and range of pH for HCV polymerase activity

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 best concentration of poly A and range of pH for reconstituting HCV polymerase activity. Methods The polymerase fragment was constructed in the plasmid pET-30a and expressed in E. coll. Results The HCV NS5b gene was expressed as a complete protein at a high level. The molecular weight of the expressed product is 65kDa within the range of expectation. The purified protein had RNA polymerase activity, which was confirmed by an ELISA test. The best concentration of the polyA and range of pH were studied in order to establish the HCV polymerase molecular model in vitro with the purified recombinant HCV NS5b protein. Conclusion The favoricte conditions for reconstituting the HCV NS56 polymerase activicty were 0. 2 μg/50μl of poly A and pH 7.7. The NS5b protein and its polymerase activity assay could be valuable in discovery of anti-HCV drugs.