摘要
目的 研究建立细胞外丙型肝炎病毒聚合酶复制模型使用的生物素标记UTP浓度及温度值 ,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 ,从我国HCVRNA阳性病人血清中扩增出HCVNS5bRNA聚合酶全长基因序列 ,构建原核表达载体pET 30a NS5b。在多个载体中选取pET 30a作为表达载体 ,选择诱导表达条件。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 96孔DNA BAND培养板 ,建立生物素标记检测HCVNS5b聚合酶活性的模型。结果 测序及读码框架分析结果表明 ,已得到相关HCVNS5bRNA聚合酶全基因序列。在最佳表达条件下 ,诱导表达的融合蛋白 (相对分子质量6 5 0 0 0 ) ,纯度大于 92 .83%HCV聚合酶活性集团完整。建立了细胞外丙型肝炎病毒聚合酶复制模型使用的最佳生物素标记UTP浓度及温度值。结论 HCV聚合酶得到高效表达和有效纯化 ,以及HCV聚合酶作用模板及温度的最佳条件分别为 0 .5mmol/L及 37℃。
Objective To explore the optimal Bio UTP concentration and temperature for establishing the replication model of HCV polymerase molecule in vitro.Methods Amplify the full length of HCV NS5b RNA polymerase gene from the sera of HCV RNA positive patients by RT PCR for the construction of a recombinant plasmid pET 30a NS5b and expression of the gene in E.coli. The expressed product was purified by Ni 2+ chelate affinity chromatography and subject to study on the conditions for degeneration and renaturation.Detect the activity of HCV NS5b RNA polymerase with Bio UTP using a 96 well DNA BAND plate.Results Sequencing and analysis of open read frame proved that the full length of HCV NS5b RNA polymerase gene was amplified.The optimal Bio UTP concentration and temperature for the establishment of replication model of HCV polymerase molecule in vitro were 0.5 mmol/L and 37℃ respectively.Under the optimal condition,the polymerase protein,with a relative molecular weight of 65 000 and a purity of above 92.83%,was expressed.Conclusion The optimal Bio UTP concentration and temperature for establishing the replication model of HCV polymerase molecule in vitro were determined,and the polymerase was highly expressed and purified.
出处
《中国生物制品学杂志》
CAS
CSCD
2004年第2期87-89,92,共4页
Chinese Journal of Biologicals
基金
"八六三"科技项目 批准号为 2 0 0 12 3 40 2 1
