应用抑制性消减杂交技术筛选HBV DNA聚合酶中RNase H的反式调节基因  

Suppression subtractive hybridization for cloning of genes transactivated by RNase H protein of HBV DNA polymerase

作　　者：王春花[1] 郎振为[1] 成军[2] 吴煜[2] 杨艳杰[2] 张黎颖[2] 党晓燕[2] 

机构地区：[1]首都医科大学北京佑安医院病理科,北京市100054 [2]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7期1564-1568,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军队十五科技攻关项目;No.01MB135~~

摘　　要：目的:应用抑制性消减杂交(SSH)技术构建HBV DNA聚合酶(DNA P)末端蛋白反式激活基因. 方法:以RNase H表达质粒pcDNA3.1(-)-RNase H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,提取mRNA并逆转录为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扩增后进行测序及同源性分析. 结果:文库扩增后得到38个白色克隆,经菌落PCR分析, 得到36个200-1 000 bp插入片段.对所得片段测序,并进行同源性分析,显示33种已知基因编码蛋白和3种未知功能基因序列,可能是RNase H反式激活靶基因, 结论:成功构建HBV RNase H反式激活基因差异表达的cDNA消减文库.AIM: To construct a subtractive cDNA library of genes transactivated by RNase H protein of hepatitis B virus (HBV) DNA polymerase with suppression subtractive hybridization (SSH) technique. METHODS: The mRNA was isolated from HepG2 cells trans-fected with pcDNA3.1(-)-RNase H and pcDNA3.1(-) empty vector, respectively, and then cDNA was synthesized. After restriction enzyme Rsal digestion, small sizes cDNAs were obtained. 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 twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up a 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 amplified library contained 38 positive clones. Colony PCR showed that 36 clones contained 200-1000 bp inserts. Sequence analysis suggested that 33 kinds of known and three kinds of novel cDNA sequences were the target genes transactivated by RNase H protein of HBV DNA P.

关 键 词：抑制性消减杂交技术 筛选 HBVDNA聚合酶 RNaseH 反式调节基因 大肠杆菌 

分 类 号：R378.21[医药卫生—病原生物学]