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目的筛选并克隆人肝细胞cDNA文库中与丙型肝炎病毒(HCV)非结构蛋白4A(NS4A) 相互作用蛋白的基因,明确其具体作用机制。方法应用酵母双杂交系统3,将聚合酶链反应法扩增的HCV NS4A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-半乳糖上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠杆菌,接种在氨苄青霉素-LB平板上,选择生长菌落,提取质粒酶切鉴定,测序并在GenBank中进行生物信息学分析。结果成功克隆出HCV NS4A基因, 构建表达载体并在酵母细胞中表达,与肝文库配合后选出既能在四缺培养基又能在铺有X-α-半乳糖的四缺培养基上生长,并变成蓝色的真阳性菌落22个,序列分析显示,筛选到的肝细胞蛋白编码基因参与细胞能量代谢、蛋白翻译合成等多种生物学过程。结论成功克隆出HCV NS4A蛋白在肝细胞内的结合蛋白, 为进一步研究NS4A蛋白的功能、阐明HCV致病的分子生物学机制提供了新线索。
Objective To screen and clone the gene of protein interacting with hepatitis C virus (HCV) nonstructural protein 4A (NS4A) in human hepatocyte cDNA library and clarify its specific mechanism of action. Methods The yeast two-hybrid system was used. 3. The HCV NS4A gene amplified by polymerase chain reaction was ligated into the yeast expression vector pGBKT7 to construct the bait plasmid. The bait plasmid was transformed into AH109 yeast cell and expressed in E. coli. pACT2 yeast cells Y187 were combined on auxotrophy medium and X-α-galactose double screening positive colonies, positive yeast colony extract plasmid was transformed into E. coli, inoculated on ampicillin-LB plate, select the growth of colonies , Extract the plasmid digestion identification, sequencing and bioinformatics analysis in GenBank. Results The HCV NS4A gene was successfully cloned and constructed. The expression vector was constructed and expressed in yeast cells. After mating with the liver library, it was selected to grow on both four-deficient medium and four-deficient medium with X-α-galactose , And turned into 22 true-positive colonies of blue. Sequence analysis showed that the selected hepatocyte protein genes involved in many biological processes such as cell energy metabolism, protein translation and synthesis. Conclusion The cloned HCV NS4A protein in hepatocytes was successfully cloned, which provided new clues for further studying the function of NS4A protein and elucidating the molecular mechanism of HCV pathology.