应用抑制性消减杂交技术克隆和筛选HBV XTP12蛋白反式激活基因的上调基因  

Cloning and screening of the up-regulated target genes transactivated by XTP12 protein of HBV by suppression subtractive hybridization

作　　者：宫嫚[1] 成军[2] 刘妍[1] 纪冬[1] 李筠[1] 

机构地区：[1]解放军第三○二医院,北京100039 [2]北京市地坛医院,北京100011

出　　处：《传染病信息》2008年第5期298-300,共3页Infectious Disease Information

基　　金：北京市自然科学基金(5042024)

摘　　要：目的克隆并筛选乙型肝炎病毒(HBV)反式激活基因XTP12的上调基因,了解其可能存在的调节功能。方法应用抑制性消减杂交技术克隆并筛选XTP12反式激活的新型靶基因。以XTP12表达质粒pcDNA3.1(-)-XTP12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RsaⅠ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XTP12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200～1 000bp插入片段。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28种编码基因,其中26种为已知基因编码蛋白,2种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、免疫及肿瘤等密切相关的蛋白编码基因,推测了XTP12可能存在的调控机制的线索。Objective To clone and screen the up-regulated target genes transactivated by XTP12 protein of HBV and explore their regulatory function. Methods Suppression subtractive hybridization（SSH） was used to clone and screen the target genes transactivated by XTP12 protein, pcDNA3.1 （-）-XTP12 was transfeeted into HepG2 cells, with peDNA3.1 （-） empty vector as control, mRNA was isolated and converted into cDNA. After restriction enzyme Rsa I digestion, tester eDNA 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 DHS. The eDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by XTP12 was constructed successfully. The amplified library contained 68 positive clones. Colony PCR showed that these clones contained 200-1 000 bp inserts. Sequence analysis was performed in 28 clones randomly, and the full length sequences were obtained with bioinformatics method. Altogether 28 coding sequences were obtained, of which 26 were known protein coding genes and 2 unknown ones. Conclusion The obtained sequences may be target genes transactivated by XTP12 among which some protein coding genes are involved in cell cycle regulation, metabolism, immunity and formation mechanism of tumor. This finding brings some new clues for studying the biological function of XTP12.

关 键 词：乙型肝炎病毒 X蛋白 反式激活 抑制性消减杂交 

分 类 号：R512.62[医药卫生—内科学]