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目的为探讨丙型肝炎病毒(HCV)F蛋白反式激活蛋白2(HCVFTP2)的功能,在真核生物酵母细胞中表达HCVFTP2基因。方法以HepG2细胞来源的mRNA作为模板,经过逆转录聚合酶链反应(RTPCR)扩增HCVFTP2基因,克隆到pGEMT载体中,双酶切后回收连接到酵母表达质粒pGBKT7中表达。提取酵母蛋白质,进行十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)和Westernblot免疫印迹分析。结果成功构建HCVFTP2基因酵母表达载体,Westernblot免疫印迹显示HCVFTP2基因在酵母细胞中表达成功。表达产物相对分子质量27kD。结论HCVFTP2在酵母中表达成功。
Objective To investigate the function of hepatitis C virus (HCV) F protein transactivator 2 (HCVFTP2) and to express HCVFTP2 gene in eukaryotic yeast cells. Methods HCVFTP2 gene was amplified by reverse transcriptase polymerase chain reaction (RTPCR) using HepG2 cell-derived mRNA as a template and cloned into pGEMT vector. The double-digested HCVFTP2 gene was ligated into yeast expression plasmid pGBKT7. Yeast proteins were extracted and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and western blot analysis. Results The yeast expression vector of HCVFTP2 gene was successfully constructed. Western blot analysis showed that HCVFTP2 gene was successfully expressed in yeast cells. Expression product relative molecular mass 27kD. Conclusion HCVFTP2 was successfully expressed in yeast.