应用抑制性消减杂交技术克隆和筛选HCVp7蛋白的反式调节基因

Screening and cloning of target genes transactivated by hepatitis C virus p7 protein

目的:构建丙型肝炎病毒HCV p7蛋白反式激活相关基因差异表达的差异cDNA,克隆HCV p7蛋白反式激活相关基因. 方法:以HCV p7表达质粒pcDNA3.1(-)-p7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Rsa Ⅰ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后进行测序及同源性分析. 结果:成功构建人HCV p7蛋白反式激活相关基因差异表达的cDNA.扩增后得到56个200-1 000 bp插入片段的克隆, 随机挑选其中33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15种编码基因,其中1个为未知功能的新基因. 结论:筛选到的cDNA全长序列,包括与细胞生长调节、物质代谢、和细胞凋亡密切相关的一些蛋白编码基因.

AIM: To clone and identify human genes transactivated by hepatitis C virus p7 (HCVp7) protein. METHODS: Suppression subtractive hybridization (SSH) and bioinformatic techniques were used for screening and cloning of the target genes transactivated by HCVp7 protein. The mRNA was isolated from HepG2 cells trans fected with pcDNA3.1(-)-p7 and pcDNA3.1(-) empty vector respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa I, cDNAs of small size were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and 2 respectively. After tester cDNA was hybridized with driver cDNA twice and underwent nested PCR twice, and then the product was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out after transfected with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive library of genes transactivated by HCVp7 was constructed successfully. The amplified library contained 71 positive clones. Colony PCR showed that 56 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 33 clones randomly, and the full-length sequences were obtained with bioinformatics method. Altogether 15 coding sequences were obtained, including 14 known and 1 unknown. CONCLUSION: The obtained sequences may be target genes transactivated by HCV p7, and some genes coding proteins get involved in cell cycle regulation, metabolism, and cell apoptosis.