应用抑制性消减杂交技术筛选乙型肝炎病毒核心抗原反式调节基因  

Screening and identification of the target genes transactivated by hepatitis B virus core antigen

作　　者：徐志强[1] 张鸿飞[1] 成军[1] 王建军[1] 刘妍[1] 纪冬[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心全军病毒性肝炎防治研究重点实验室,北京市100039

出　　处：《世界华人消化杂志》2004年第11期2576-2580,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金攻关项目;No.C03011402;No.C30070689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~~

摘　　要：目的:筛选与克隆HBcAg反式调节基因,了解其在体内的调节功能线索及机制. 方法:以分子生物学技术构建HBcAg的真核表达载体pcDNA3.1(-)-HBcAg,以毒乏达质粒pcDNA3.1(-)-HBcAg转染HepG2细胞,以空载体pcDN.A3.1(-)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扩增后进行测序及同源性分析. 结果:成功构建人HacAg激活基因差异表达的cDNA消减文库.文库扩增后得到33个阳性克隆,进行菌落PCR分析,均得到206-800 bp插入片段.对插入片段测序,并通过生物信息学分析获得其全长基因序列,结果共获得17种编码基因. 结论:筛选到的cDNA全长序列,包括一些与细胞生长调节、物质代谢及肿瘤发生密切相关的蛋白编码基因,推测了HBcAg在体内可能存在的调控机制的线索,尚需进一步的实验证明.AIM: To clone and identify human genes transactivated by hepatitis B virus core antigen (HBcAg) using suppression subtractive hybridization (SSH) technique. METHODS: SSH and bioinformatic techniques were used for screening and cloning of the target genes transactivated by HBcAg protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-HBcAg 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 fragments of cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA and underwent nested PCR twice, the product was suBcloned into T/A plasmid vectors to set up the subtractive library. Amplifi cation of the library was carried out with eE.colistrain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive library of genes transactivated by HBcAg was constructed successfully. The amplified library contains 33 positive clones. Colony PCR shows that these clones contain 200-800 bp inserts. The full-length sequences were obtained with bioinformatics method. Altogether 17 coding sequences were identified. CONCLUSION: The obtained sequences may be the target genes transactivated by HBcAg, among which some genes are involved in cell cycle regulation, metabolism, and tumor immunity and development.

关 键 词：抑制性消减杂交技术 筛选 乙型肝炎病毒 核心抗原 反式调节基因 

分 类 号：R373.21[医药卫生—病原生物学]