丙型肝炎病毒非结构蛋白NS5A反式激活基因的克隆化研究

SUPPRESSION SUBTRACTIVE HYBRIDIZATION FOR CLONING OF GENES TRANSACTIVATED BY NS5A PROTEIN OF HEPATITIS C VIRUS

应用抑制性消减杂交技术构建丙型肝炎病毒非结构蛋白 5A(HCVNS5A)反式激活基因差异表达的cDNA消减文库 ,克隆HCVNS5A蛋白反式激活相关基因。以HCVNS5A表达质粒pcDNA3 1(- ) NS5A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 ,经RsaI酶切后 ,将实验组cDNA分成两组 ,分别与两种不同的接头衔接 ,再与对照组cDNA进行两次消减杂交及两次抑制性PCR ,将产物与T/A载体连接 ,构建cDNA消减文库 ,并转染大肠杆菌进行文库扩增 ,随机挑选克隆PCR扩增后进行测序及同源性分析。文库扩增后得到 12 1个阳性克隆 ,经菌落PCR分析 ,得到 115个 2 0 0～10 0 0bp的插入片段 ,对其中的 90个片段测序 ,并进行同源性分析 ,显示 31种已知基因编码蛋白和 15种未知功能基因序列 ,包括一些与细胞周期、细胞凋亡、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NS5A反式激活靶基因。结果提示 ,成功构建了HCVNS5A反式激活基因差异表达的cDNA消减文库 ,该文库的建立为进一步阐明HCVNS5A反式调节的靶基因及致肝细胞癌发生的分子生物学机制提供了理论依据。

To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid 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. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins