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为克隆与丙型肝炎病毒 (HCV)NS3蛋白结合的肝细胞中的蛋白基因 ,采用酵母双杂交系统Ⅲ技术进行筛选。构建HCVNS3蛋白诱饵酵母质粒 ,转化酵母AH10 9后与含文库质粒的酵母Y187进行配合 ,在营养缺陷培养基上进行双杂交筛选。选择既能在 4重营养缺陷培养基(SD/ Trp Leu Ade His)上生长 ,又能在涂有X α 半乳糖(X α gal)的四缺培养平皿上生长的蓝色菌落 ,提取此酵母克隆的质粒 ,转化大肠杆菌后进行测序 ,并进行生物信息学分析。分析的结果显示 ,筛选出 19个阳性克隆 ,包括已知功能基因 17个 ,还有 1个克隆的测序结果与GenBank中的 1个未知功能序列有 4 4 %的同源性 ,初步证明了此基因与HCVNS3有结合活性。说明成功克隆了HCVNS3蛋白结合蛋白基因 ,为以后研究此基因在HCV致病机制以及在肝细胞中的生理功能提供了线索。
To clone protein genes in hepatocytes that bind to the Hepatitis C virus (HCV) NS3 protein, the yeast two-hybrid system III technique was used for screening. The bait yeast plasmids of HCVNS3 protein were constructed and transformed into yeast AH109. The yeast plasmids were combined with yeast Y187 containing library plasmids to screen for two hybrids on auxotrophic medium. The blue colonies that grew on quadruple auxotrophic medium (SD / Trp Leu Ade His) and on four-deficient culture plate coated with X? Gal were selected and the yeast The cloned plasmid was transformed into E. coli and sequenced. Bioinformatic analysis was also carried out. The results of the analysis showed that 19 positive clones were screened out, including 17 known functional genes, and another one had 4 4% homology with one unknown functional sequence in GenBank. The gene has a binding activity to HCV NS3. The successful cloning of the HCV NS3 protein binding protein gene provides a clue for the future study of this gene in the pathogenesis of HCV and in liver cells physiological function.