丙型肝炎病毒核心蛋白结合蛋白6基因转染肝癌细胞的基因表达谱芯片分析

Gene expression profile of HepG2 cell transfected with hepatitis C virus core protein-binding protein 6 gene

目的:筛选并克隆丙型肝炎病毒(HCV)核心蛋白的肝细胞结合蛋白基因,并对新基因转染肝癌细胞的基因表达谱进行分析,探索该基因表达对肝细胞基因表达的调节机制。方法:应用酵母双杂交技术,以HCV的核心蛋白作为“诱饵(bait)”,筛选鉴定与其结合的肝细胞中蛋白的编码基因。应用生物信息学(bioinformatics)技术,分析其中筛选得到的人HCV核心蛋白结合蛋白6(HCBP6)基因的全长编码序列,并构建HCBP6基因的真核表达载体pcDNA3.1(-)-HCBP6。应用基因表达谱芯片技术对重组表达质粒pcDNA3.1(-)-HCBP6转染的HepG2细胞和空载体处理的相同细胞差异表达的mRNA进行检测。结果:通过酵母双杂交技术的筛选和鉴定,结合生物信息学分析,确定人的HCBP6基因由456 nt组成,编码152 aa的蛋白。基因表达谱芯片所检测的1 152条目的基因均为GenBank中登录的基因,HCBP6表达质粒转染的细胞有20条差异表达基因,其中13条基因表达增强,7条基因表达降低。这些差异表达的基因与细胞信号转导、增生、分化及生长调节密切相关。结论:酵母双杂交技术结合生物信息学技术,是克隆蛋白结合蛋白的有效方法,基因表达谱芯片技术对于初步全面探索新基因的功能提供重要的资料。本实验结果为进一步阐明HCV核心蛋白与Hcbp6相互作用后的肝细胞生物大分子变化提供了理论依据。

AIM:TO screen and clone genes of hepatic protein interacting with hepatitis C virus (HCV) core protein and analyze the gene expression profiles of HepG2 cell transfected with HCV core protein-binding protein 6 (HCBP6) gene. METHODS:Using yeast two-hybrid system 3, bait plasmid of HCV core protein was constructed and genes encoding HCV core protein-binding protein were screened and identified. One gene named HCBP6 was identified. Human full-length encoding gene of HCBP6 and its amino acid sequences were identified by bioinformatics method, and the recombined expression plasmid pcDNA3.1(-)-HCBP6 was constructed. mRNA from HepG2 cells transfected with pcDNA3.1(-)-HCBP6 and the empty vector were detected with cDNA microarray, respectively. RESULTS:Human HCBP6 cDNA sequence was identified. The coding sequence for human HCBP6 consists of 456 nt and its protein consists of 152 aa. Twenty of 1152 genes obtained from gene expression profile analysis differed from those in GenBank, in which 13 genes were up-regulated and 7 genes were down-regulated in HepG2 cells transfected with HCBP6 plasmid. These genes differentially regulated by HCBP6 protein included human genes encoding proteins involved in signal transduction, cell proliferation, differentiation, and growth regulation. CONCLUSION:The bioinformatics combined yeast two hybrid technique is a powerful method for screening and analysis of genes of hepatic protein interacting with HCV core protein. The findings obtained by cDNA microarray technique provided significant data for preliminary understanding of the biological function of new gene, and also provided some dues for furbher clarifying the molecular biological processes of hepatocytes in interaction between HCV core protein and HCBP6.