丙型肝炎病毒核心蛋白结合蛋白6的反式调节基因研究

The study on genes trans-regulated by hepatitis C virus core protein-binding protein 6

目的:筛选、克隆新基因HCBP6转染肝癌细胞后的反式调节基因,探索HCBP6基因表达对肝细胞基 因表达谱的影响。方法:应用酵母双杂交技术和生物信息学(bioinformatics)技术,筛选得到的人HCV核心蛋白结 合蛋白6(HCBP6)的基因。构建HCBP6基因的真核表达载体pcDNA3.1( ) HCBP6,应用抑制性消减杂交技术对 重组表达质粒pcDNA3.1( ) HCBP6转染的HepG2细胞和空载体转染的相同细胞差异表达的mRNA进行研究,将 富集的二次PCR产物与T/A载体连接,并转化大肠杆菌进行文库扩增,随机挑取克隆聚合酶链反应(PCR)扩增后 进行测序及同源性分析。结果:消减文库扩增后得到30个阳性克隆,菌落PCR分析显示,其中24个克隆含有200 ～1000bp插入片段。对插入片段测序,并通过生物信息学分析,结果共获得11种蛋白编码基因。结论:应用SSH 技术成功筛选了HCBP6对肝癌细胞的反式调节基因,本实验结果对初步探索新基因的功能提供重要的信息,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。

Objective:To analyze the expression profile of genes trans-regulated by HCV core protein-binding protein 6 (HCBP6) gene.Methods:Human HCBP6 full-length encoding gene and its amino acid sequences was identified using bioinformatics method and the recombined expression plasmid pcDNA3.1(-)-HCBP6 was constructed. Suppression subtractive hybridization (SSH) technique was employed to detect the mRNA from HepG2 cells transfected with pcDNA3.1(-)-HCBP6 and the empty vector, respectively. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results:According to yeast two-hybrid screening and the bioinformatics analysis results, human HCBP6 cDNA sequence was identified. The encoding sequence for human HCBP6 consists of 456 nt and its protein consists of 152 aa. The amplified subtractive library contains 30 positive clones. Colony PCR analysis shows there are 24 clones containing 200-1 000 bp inserts. Sequence analysis was performed and 11 kinds of encoding sequences were achieved. Conclusion:The bioinformatics combined yeast two hybrid methods is a powerful method for the screening and analysis of genes of hepatocyte protein interacting with HCV core protein. The findings obtained using SSH provide significant data for preliminary understanding the biological function of a new identified gene.