丙型肝炎病毒非结构蛋白NS5B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非结构蛋白5B（HCV NS5B）转染细胞差异表达cDNA消减文库，克隆HCV NS5B蛋白反式激活相关基因。方法：以HCV NS5B表达质粒pcDNA3.1（-）-NS5B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（SSH）分析。随机挑取克隆进行测序及同源性分析。结果：文库扩增后得到35个阳性克隆，经菌落PCR分析显示其中26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16种已知基因编码蛋白和1种未知功能基因序列，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NS5B反式激活靶基因。结论：成功构建了HCV NS5B反式激活基因差异表达的cDNA消减文库，为进一步阐明HCV NS5B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 5B of hepatitis C virus

Objective:To construct a subtractive cDNA library of genes transactivated by NS5B protein of hepatitis C virus with suppression subtractive hybridization technique(SSH).Methods:The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-NS5B 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 cDNA was sequenced and analyzed in GenBank with Blast search after the PCR amplification of the library.Results:The amplified library contains 35 positive clones.(Colony) PCR shows that 26 clones contain 200-1000 bp inserts.Sequence analysis was performed in 26 clones,and the full length sequences were obtained with bioinformatics method.Altogether 17 kinds of coding sequences were achieved,which consisted of 16 kinds of known and one unknown gene.The obtained sequences may be target genes transactivated by NS5B protein of HCV,(among) which some genes coding proteins involved in cell cycle regulation,cell signal transduction pathway and tumour develo(pment).Conclusions:A subtractive library of genes transactivated by NS5B protein of HCV was constructed successfully,which brought some new clues for studying the biological functions and pathogenesis of the viral proteins.hepatitis C-like virus;viral nonstructural proteins;transactivation(genetics)