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目的 克隆与丙型肝炎病毒 (HCV)核心蛋白结合的肝细胞中的未知蛋白基因。方法 应用酵母双杂交系统 ,构建丙型肝炎病毒核心蛋白诱饵质粒 ,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。筛选出 30个克隆 ,既能在四缺营养缺陷培养基上生长 (SD Trp Leu Ade His)又能在涂有x α gal的四缺培养平皿上长出蓝色菌落 ,提取此酵母克隆的质粒 ,转化大肠埃希菌后进行测序 ,进行生物信息学分析。结果 分析结果显示 ,其中 6号克隆的测序结果与Genbank中的 2个未知功能序列有 98%的同源性 ,初步证明了此基因与丙型肝炎病毒核心蛋白有结合性。结论 丙型肝炎病毒核心蛋白结合蛋白肝基因克隆成功。
Objective To clone an unknown protein gene in hepatocytes that binds to the hepatitis C virus (HCV) core protein. Methods The yeast two-hybrid system was used to construct the bait plasmids of hepatitis C virus core protein. The transformed bait plasmids were transformed into yeast AH10 9 and then ligated with yeast Y187 containing the library plasmid. Two-hybrid screening was carried out on auxotrophic medium. Thirty clones were screened for growth on both SD Trp Leu Ade His and blue colonies on quadruplicate plated plates coated with x α gal, and the yeast cloned plasmids were extracted , Transformed into Escherichia coli after sequencing, bioinformatics analysis. The result of analysis of the results showed that the sequence of No. 6 clone had 98% homology with two unknown functional sequences in Genbank, and it was preliminarily proved that this gene had the binding with hepatitis C virus core protein. Conclusion Hepatitis C virus core protein binding protein gene was cloned successfully.