应用噬菌体展示技术筛选丙型肝炎病毒核心蛋白反式激活基因HCTP4启动子DNA的结合蛋白

Screening of Promoter Binding Protein of HCTP4 Transactivated by Hepatitis C Virus Core Protein from Human Liver cDNA Library by Phage Display

目的:筛选丙型肝炎病毒(HCV)核心蛋白反式激活基因HCTP4启动子DNA结合蛋白,探索HCTP4基因的表达调控机制。方法:应用噬菌体表面展示技术,以多聚酶链反应(PCR)技术扩增的HCTP4启动子DNA片段作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行4轮“吸附-洗脱-扩增”富集过程,噬斑裂解液PCR扩增后,回收产物并进行克隆化,对所筛选克隆进行DNA序列分析和同源性搜索。结果:噬菌体经富集后,筛选出12个阳性克隆,成功构建了克隆载体。序列测定后经过同源性搜索,确定了与HCV核心蛋白反式激活基因HCTP4启动子DNA结合的蛋白有:核糖体蛋白L15、视网膜母细胞瘤结合蛋白8。结论:用噬菌体人肝cDNA文库筛选得到HCV核心蛋白反式激活基因HCTP4启动子DNA结合蛋白,分析了这些蛋白的功能,为研究HCTP4的生物学调节机制,进一步阐明HCV核心蛋白的致病机制奠定了基础。

Objective: To screen the promoter binding protein of HCTP4 transactivated by the core protein of hepatitis C virus from human liver cDNA library and investigate the expression and regulation mechanism of the new gene HCTP4.Methods: The promoter sequence of HCTP4 was amplified by polymerase chain reaction (PCR) as selected molecule, the T7 select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque were performed to amplify and PCR amplified DNA fragments were cloned into pGEM-Teasy vectors, 12 positive plaque were chosen for DNA sequencing.Results: After BLAST in all positive clones, we found that the human ribosomal protein L15 and human retinoblastoma 8 binding protein are HCTP4 promoter binding proteins.Conclusion: HCV core protein transactivate gene-HCTP4 promoter binding proteins has been screened.The results will pay the way for the study of the molecular mechanism of HCTP4 gene expression and regulation.