【摘要】 目的构建HCV聚合酶基因的重组原核表达质粒后,研究了获得高效表达聚合酶蛋白的最适条件,同时摸索表达产物的纯化方法以及最佳变性和复性条件,研究建立细胞外丙型肝炎病毒聚合酶复制模型使用的HCV聚合酶作用模板及酶浓度的最佳浓度,为筛选药物创造条件。方法使用高保真PfuDNA聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性病人血清中扩增出HCV NS5b RNA聚合酶全长基因序列,构建了原核表达载体pET30aNS5b等。在多个载体中选取pET-30a作为表达载体,选择各种诱导表达条件。进一步利用Ni2十金属离子螯和亲和层析将其纯化,对该酶蛋白的变性和复性条件进行研究。利用DNA-BAND培养板96孔板,建立生物素标记检测HCV NS5b聚合酶活性的细胞外分子模型。同时,摸索了聚合酶作用模板以及酶浓度对聚合酶模型的影响的相关条件。结果测序及读码框架分析结果表明得到相关HCV NS5b RNA聚合酶全基因序列,在最佳表达条件下,诱导表达融合蛋白(65×103),得到了纯度大于92.83%的酶蛋白,研究确定了HCV聚合酶作用模板及酶浓度的最佳条件。结论HCV聚合酶的高效表达和有效纯化,以及HCV聚合酶作用模板及酶浓度的最佳条件研究,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。更多还原

【Abstract】 Objective To study the best requirement of the HCV polymerase primer and enzyme concentration,and to establish the HCV polymerase molecular model in vitro.Methods RT-PCR method was used to amplify HCV NS5b poly-merase gene from a HCV(+) Chinese patient′s serum.The polymerase fragment was constructed in the plasmid pET-30a and expressed in Ecoli.The NS5b protein was purified by immobilized metal affinity chromatography(IMAC).Meantime the best prerequisite of the HCV polymerase enzyme primer and concentration for viral replication and pathogenesis were studied.Results The HCV NS5b gene was expressed at a high level.The molecular weight of the expressed product was 65 kDa within the range of our expectation.The purified protein had shown to possess RNA polymerase activity in the assay using ELISA.The best factor of the HCV polymerase enzyme primer and concentration was found.Conclusion The NS5b protein and the method of NS5b polymerase activity assay could be a valuable model in discovery of anti-HCV drugs.更多还原