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目的:用酵母双杂交技术筛选肝细胞中与HCV E1蛋白结合蛋白的编码基因. 方法:用多聚酶链反应(PCR)法扩增HCV E1基因,连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落,增菌后提出质粒,转化入大肠杆菌(DH5α),提出质粒并测序, 进行生物信息学分析. 结果:获得了19个与E1蛋白特异陛结合的阳性克隆,其中16个克隆为已知蛋白基因和3个克隆为未知功能蛋白基因. 结论:成功克隆出与丙型肝炎病毒E1蛋白结合的肝细胞蛋白,为进一步研究HCV E1在HCV致病中的作用提供了新线索.
OBJECTIVE: To screen HCV E1 protein-binding protein coding genes in hepatocytes by yeast two-hybrid technique.Methods: The HCV E1 gene was amplified by polymerase chain reaction (PCR) and inserted into the yeast expression vector pGBKT7 to construct the bait plasmid. Cells AH109 were expressed in and then mixed with the yeast cell Y187 transformed with the human liver cDNA library plasmid pACT2 to perform dual screening positive on auxotrophy medium and X-α-galactose (X-α-gal) The colony was added with plasmid and transformed into Escherichia coli DH5α.The plasmid was cloned and sequenced for bioinformatics analysis.Results: Nineteen positive clones were obtained, of which 16 were known Protein gene and three clones were unknown functional protein.Conclusion: The successful cloning of hepatocyte protein that binds to hepatitis C virus E1 protein provides new clues for further study on the role of HCV E1 in HCV pathogenesis.