【摘要】 在大肠杆菌菌株BL21(DE3)中表达并纯化HCV的依赖于RNA的RNA多聚酶(RNAdependentRNApolymerase,RdRp,NS5B蛋白)。以HCV正、负链RNA3′末端的序列为模板,体外研究NS5B蛋白催化的RNA合成。结果显示,正链RNA在体外不能指导RNA合成,而负链RNA模板可以产生一条全长的正链RNA产物,表明NS5B对负链RNA具有模板特异性。NS5B对负链RNA的特异性在模板竞争性实验中得到进一步证实,正链RNA的存在和竞争对以负链为模板的RNA合成没有影响。这样,就合理解释了在HCVRNA复制时正链RNA的数量远比负链RNA多这一问题。同时,本实验的结果也为进一步研究病毒或其它细胞因子参与以正链RNA为模板进行的RNA合成,以及有关负链RNA模板特性的研究奠定了基础。更多还原

【Abstract】 A recombinant HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) was expressed in E. coli strain BL21(DE3). The purified protein was used to investigate the RNA syntheses from the 3′ terminal sequences of HCV positive and negative strand RNA in vitro. The positive strand RNA did not direct the synthesis of negative strand RNA; however, the negative-strand RNA was able to generate a full-length positive strand RNA. These results demonstrate that NS5B has the template specificity on negative strand RNA. The template specificity was further confirmed by template competition assay. The competition of positive-strand RNA did not affect the RNA synthesis from the negative strand RNA. The template specificity of NS5B provides a reasonable explanation for the excesses production of positive over negative strand RNA during the HCV genome replication. In addition, these findings provide some clues to further work. The research on viral or cellular factors involved in the RNA synthesis from positive-strand RNA, as well as the features of negative-strand RNA template will lead to the better understanding of HCV replication.更多还原