应用抑制性消减杂交技术克隆HCV NS3蛋白反式激活基因2的上调基因

Screening and cloning of the target genes transactivated by human gene 2 transactivated by hepatitis C virus NS3 protein using suppression subtractive hybridization

目的:应用抑制性消减杂交(SSH)技术构建丙型肝炎病毒(HCV)NS3蛋白反式激活相关基因差异表达的cDNA消文库,克隆HCV NS3蛋白反式激活相关基因. 方法:以HCV NS3表达质粒pcDNA3.1(-)-NS3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 NS3蛋白反式激活相关基因差异表达的cDNA消减文库.文库扩增后得到61个白色克隆,进行菌落PCR分析,均得到100-1000 bp插入片段.挑取30 个插入片段测序分析,得到30个已知功能基因序列. 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、免疫及细胞凋亡密切相关的蛋白编码基因,推测了NS3TP2可能存在的调控机制的线索.

AIM: To clone and identify human genes transactivated by NS3TP2 by constructing a cDNA subtractive library with suppression subtractive hybridization technique. METHODS: Suppression subtractive hybridization (SSH) and bioinformatics were used for screening and cloning of the target genes transactivated by NS3TP2 protein. The mRNA was isolated from HepG2 cells transfected pcDNA3.1 (-)-NS3TP2 and pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then 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 was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive library of genes transactivated by NS3TP2 was constructed successfully. The amplified library contained 61 positive clones. Colony PCR showed that these clones contained 200-1000 bp inserts. Sequence analysis was performed in 30 clones, and the full length sequences were obtained with bioinformatics method. Altogether 21 coding sequences were identified. CONCLUSION: The obtained sequences may be target genes transactivated by NS3TP2, among which some genes coding proteins involve cell cycle regulation, metabolism, immunity and cell apoptosis. Advanced experiments need to be done to prove this finding.