丙型肝炎病毒非结构蛋白NS4B基因酵母表达载体的构建及表达

Cloning and expression of non-structural protein 4B gene of hepatitis C virus in yeast

目的:构建丙型肝炎病毒(HCV)非结构蛋白NS4B基因的酵母细胞表达载体,为探索HCVNS4B在细胞内的相互作用蛋白奠定基础。方法:用多聚酶链反应(PCR)的方法在HCV全长质粒pBRTM/HCV-1为模板扩增HCVNS4B基因,克隆到pGEM-T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(SDS-PAGE)和Western免疫印迹分析。结果:成功构建HCVNS4B基因酵母表达载体,Western免疫印迹分析显示了HCVNS4B在酵母细胞中融合表达。表达产物在胞内存在,融合蛋白的相对分子量约为48ku左右。结论:HCVNS4B蛋白在酵母中表达成功。

Objective：To investigate the gene expression of non-structural protein 4B （NS4B） of hepatitis C virus （HCV） in yeast, nethods：Polymerase chain reaction （PCR） was performed to amplify the gene of HCV NS4B from the plasmid pBRTM/HCV-1 containing the whole fragment of HCV and the gene was cloned into pGEM T vector. The gene of HCV NS4B was cut from the recombinant pGEM T-NS4B plasmid and cloned into yeast expression plasmid pGBKT7, then pGBKT7-NS4B was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting analysis. Results： HCV NS4B gene was successfully cloned into pGBKT7. The results of SDS-PAGE and Western blotting assay indicated that the relative molecular weight of the expressed product was approximately 48 ku and HCV NS4B protein existed within yeast cells. Conclusion：The findings suggested that HCV NS4B protein was successfully expressed in yeast system.