应用抑制性消减杂交技术克隆丙型肝炎病毒核心蛋白反式激活基因

应用抑制性消减杂技术构建丙型肝炎病毒（HCV）核心蛋白反式激活基因差异表达的cDNA消减文库，克隆HCV核心蛋白反式激活相关基因。以HCV核心表达质粒pcDNA3.1(-)-core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，从中提取mRNA并逆转录为cDNA，经Rsa I酶切后将实验组cDNA分成2组，分别与2种不同的接头衔接，再与对照组cDNA进行2次消减杂交及2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。结果显示，成功构建人HCV核心蛋白反式激活基因差异表达的cDNA消减文库。文库扩增后得到233个白色克隆，进行菌落PCR分析，其中213个均得到100～1000bp插入片段。挑取63个插入片段测序分析，其中6个cDNA片段为未知序列，通过生物信息学分析获得其全长序列，已被GenBank收录。提示6个新的cDNA全长序列，可能是HCV核心蛋白反式激活靶基因。

SUPPRESSION SUBTRACTIVE HYBRIDIZATION FOR CLONING OF GENES TRANSACTIVATED BY HCV CORE PROTEIN

To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was 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 were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.