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

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。

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

Objective:To investigate the gene expression of non-structural protein 4B (NS4B) of hepatitis C virus (HCV) in yeast. Methods: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.