丙型肝炎病毒非结构蛋白NS4B反式激活基因的克隆化研究

目的应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)转染细胞差异表达cDNA消减文库,克隆HCV NS4B蛋白反式激活相关基因.方法以HCV NS4B表达质粒pcDNA3.1(-)-NS4B转染HepG2细胞,以空载体pcDNA3.1(-)为对照,制备转染后的细胞裂解液,提取mRNA并逆转录为cDNA,进行抑制性消减杂交分析.将富集的二次PCR产物与T/A载体连接,并转染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.结果文库扩增后得到33个阳性克隆,经菌落PCR分析显示其中28个克隆含有大小不等的200～1000 bp插入片段.测序及同源性分析显示,12种已知基因编码蛋白,包括一些与细胞周期、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因,可能是NS4B反式激活靶基因.结论成功构建了HCV NS4B反式激活基因差异表达的cDNA消减文库,为进一步阐明HCV NS4B反式调节的靶基因在肝炎、肝纤维化和肝细胞癌发生的分子生物学机制提供理论依据.

Cloning of genes transactivated by nonstructural protein 4B of hepatitis C virus

Objective To construct a subtractive cDNA library of genes transactivated by NS4B protein of hepatitis C virus (HCV) with suppression subtractive hybridization(SSH) technique.Methods The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-NS4B and pcDNA3.1(-) empty vector, respectively, then the cDNA was synthesized. SSH method was employed to analyze the differentially expressed RNA sequence between the two groups. 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 The amplified library contains 33 positive clones. Colony PCR shows that 28 clones contain 200-1000 bp inserts. Sequence analysis was performed in 28 clones, and the full length sequences were obtained with bioinformatics method. Altogether 12 kinds of coding sequences were achieved. The obtained sequences may be target genes transactivated by NS4B protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell signal transduction pathway and tumour development. Conclusion A subtractive library of genes transactivated by NS4B protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins.