抑制性消减杂交技术克隆和筛选丙型肝炎病毒F蛋白的反式调节基因

目的 构建丙型肝炎病毒（HCV）F蛋白反式激活相关基因差异表达的差异cDNA，克隆HCV-F蛋白反式激活相关基因。方法 以HCV-F表达质粒pcDNA3．1（-）-F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并合成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聚合酶链反应，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，随机挑选克隆聚合酶链反应后进行测序及同源性分析。结果 成功构建人HcVF蛋白反式激活相关基因差异表达的cDNA。扩增后得到56个200～1000bP插入片段的克隆，随机挑选其中28个插入片段测序，并通过生物信息学分析获得其全长基因序列，结果共获得19种编码基因，其中2个为未知功能的新基因。结论 筛选到的cDNA全长序列，包括一些与细胞生长调节、物质代谢和细胞凋亡密切相关的蛋白编码基因。

Screening and cloning target genes transactivated by hepatitis C virus F protein using suppression subtractive hybridization technique

Objective To identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique. Methods Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with 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 DH5 a . The cDNA was sequenced and analyzed in GenBank with blast search after PCR. Results The subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown. Conclusion The obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.