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由于全长丙型肝炎病毒NS5B的疏水性 ,其表达和纯化非常困难。为了分泌表达NS5B ,作者对NS5B进行截短 ,缺失其疏水部分从而在大肠杆菌细胞中分泌表达HCVNS5B基因 ,并测定其活性。用逆转录多聚酶链反应 (RT PCR)的方法 ,设计截去NS5B疏水部分 ,PCR引物以HCV全长质粒pBRTM/HCV 1为模板 ,克隆到pGEM Teasy载体中 ,双酶切后回收连接到pET 2 1b中表达。大肠杆菌培养上清过柱纯化 ,进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析 ,并用液闪近端分析 (SPA)法分析NS5BRdRp活性。成功地构建了NS5B基因大肠杆菌表达载体 ,Western免疫印迹显示NS5B蛋白在大肠杆菌细胞中表达。表达产物在大肠杆菌培养上清中存在 ,分子量 6 8kD左右 ,而且 [3 H]总掺入率达 6 90 0cpm。NS5B蛋白在大肠杆菌上清中表达成功 ,经过活性测定 ,所表达的NS5B具有活性功能
Due to the hydrophobic nature of the full-length hepatitis C virus NS5B, its expression and purification is very difficult. To secrete NS5B, the authors truncated NS5B, deleted its hydrophobic portion and secreted the HCV NS5B gene in E. coli cells and assayed its activity. The hydrophobic part of NS5B was cloned by reverse transcription polymerase chain reaction (RT PCR). The PCR primers were cloned into pGEM Teasy vector using the full-length HCV plasmid pBRTM / HCV 1 as a template. After double digestion, the PCR product was ligated into pET 2 1b. Escherichia coli culture supernatants were column-purified and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis. The NS5BRdRp activity was analyzed by liquid-flash proximal assay (SPA). The NS5B gene E. coli expression vector was successfully constructed and Western immunoblotting showed that the NS5B protein was expressed in E. coli cells. The expression product existed in E. coli culture supernatant with a molecular weight of about 68 kD, and the total incorporation rate of [3 H] was 6 90 0 cpm. The NS5B protein was successfully expressed in E. coli supernatants. After being assayed for activity, the expressed NS5B has an active function