摘要
目的 表达具有野生起点的丙型肝炎病毒 (HCV)RNA依赖的RNA聚合酶 (RdRp)并研究其聚合活性 ,分析其是否具有逆转录酶活性。方法 构建截短的具有野生起点的HCVRdRp表达质粒 ,观察不同诱导温度对表达产物可溶性的影响。以多聚腺苷为模板 ,分别观察在寡聚脱氧胸苷和寡聚尿苷引导下RNA的合成。以互补引物模板评价所表达的HCVRdRp的逆转录酶活性。 结果 在 16°C诱导温度条件下获得截短具有野生起点的HCVRdRp的可溶性表达。在聚合反应体系中同时具有引物与模板时 ,获得RNA链的合成 ,多聚核苷为引物的聚合效率明显高于以多聚脱氧核苷为引物的聚合效率。在表达的HCVRdRp指导下 ,脱氧核苷掺入到互补引物模板体系中 ,随着反应时间的延长 ,10聚的互补引物 /模板延伸为 13聚的产物 ,但不能延长至 14聚。结论 获得可溶性的HCVRdRp表达 ,具有RNA依赖的RNA聚合酶活性及有限的逆转录酶活性。其RNA依赖的RNA聚合酶活性有一定的引物依赖性。
Objective To express hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp), and analysis it activity and reverse transcription. Methods Hydrophilicity profile and hydrophobicity profile were used to analyse hydrophilicity of full-length HCV RdRp, HCV RdRp truncated at the Cterminal region and truncated HCV RdRp with his-tag tail, respectively. The impact of different inducing temperatures on solubility of protein expressed were evaluated.RNA polymerase activity was observed with primers U12 and dT15 and template poly(rA). The reverse transcription activity was evaluated by using symmetrical primer-template. Results Soluble HCV RdRp was obtained by decreasing temperature to 16 °C after induction and the proteins expressed at 20 °C and 25 °C are insoluble. Among different reaction systems, RNA polymerase activity was the highest in the reaction containing primer U12 and template poly(rA), and the reaction containing primer dT15 and template poly(rA) also showed activity compared with the reaction containing only poly(rA). With symmetrical primer-template as template and primer, HCV RdRp catalysis dNTP incorporating into the template. However, the template only elongate to 13-mer otherwise 14-mer, and also long time was required for reaction to elongate template. Conclusions Soluble HCV RdRp was obtained and the recombinant soluble protein expressed is active. Its RNA polymerase activity was dependent upon the primer in vitro with limited reverse transcription activity.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2003年第2期97-100,共4页
Chinese Journal of Infectious Diseases
基金
首都二四八工程重大创新工程资助项目 (生物医药领域 ) ( 95 5 0 2 12 70 0 )
国家"十五"攻关项目 ( 2 0 0 1B70 5B0 6 )
中华医学基金专项人才基金资助项目
