摘要
目的:筛选与克隆丙型肝炎病毒(HCV)NS3反式激活基因1 的反式激活基因,了解其可能存在的调节功能线索. 方法:应用抑制性消减杂交(SSH)技术及生物信息学(bioin- formatics),技术筛选并克隆NS3TP1反式激活的新型靶基因. 以NS3TP1表达质粒pcDNA3.1(-)-NS3TP1转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,制备转染后的细胞裂解液,提取mRNA并逆转录为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聚合酶链反应(PCR),将产物与pGEM-Teasy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:成功构建人NS3TP1反式激活基因差异表达的cDNA 消减文库.文库扩增后得到68个阳性克隆,进行菌落PCR 分析,均得到200-1000 bp插入片段.随机挑选其中36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23种编码基因,其中3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、免疫及细胞凋亡密切相关的蛋白编码基因, 推测了NS3TP1可能存在的调控机制的线索.
AIM: To clone and identify human genes transactivated by human gene 1 transactivated by hepatitis C virus NS3 protein (NS3TP1) by constructing a cDNA subtracttive library with suppression subtractive hybridization (SSH). METHODS: Suppression subtractive hybridization and bioinformatics were used for screening and cloning of the target genes transactivated by NS3TP1 protein. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-NS3TP1 and pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed cDNA 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. Tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction (PCR) twice, and then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out 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 NS3TP1 was constructed successfully. The amplified library contained 68 positive clones. Colony PCR showed that these clones contained 200-1000 bp inserts. Sequence analysis was performed in 36 clones, ramdomly, and the full length sequences were obtained with bioinformatics method. Altogether 23 coding sequences were obtained, which consisted of 20 known and 3 unknown ones. CONCLUSION: The obtained sequences may be target genes transactivated by NS3TP1. among which some genes coding proteins involve in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brings some new clues for studying the biological functions of NS3TP1.
出处
《世界华人消化杂志》
CAS
2004年第4期843-846,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.C03011402
No.C30070689
No.C39970674
No.C30371288军队"九
五"科技攻关项目
No.98D063军队回国留学人员启动基金项目
No.98H038军队"十
五"科技攻关青年基金项目
No.01Q138军队"十
五"科技攻关面上项目
No.01MB135~~
