应用抑制性消减杂交技术克隆乙型肝炎病毒DNAPTP1的反式调节基因

Cloning of hepatitis B virus DNAPTP1 transactivating genes by suppression subtractive hybridization technique

目的:应用抑制性消减杂交技术及生物信息学技术筛选并克隆乙型肝炎病毒DNAPTP1反式激活的新型靶基因．方法:以HBV DNAPTP1表达质粒pcDNA3．1(-)- DNAPTP1 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DNAPTP1反式激活基因差异表达的cDNA 消减文库．文库扩增后得到60个白色克隆,经菌落PCR分析,得到32个200-1 000 bp插入片段．对所得片段测序,并进行同源性分析,获得3个差异表达的已知蛋白基因和4个未知功能的染色体序列．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DNAPTP1可能存在的调控机制的线索．

AIM： To clone and identify the new target genes transactivated by DNAPTP1 of the hepatitis B virus with the suppression subtractive hybridization technique and bioinformatics. METHODS： The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 （-）-DNAPTP1, and then it was transcribed into the cDNA. The pcDNA3.1 （-） empty vector was used as control. After digested by Rsa I, the tester cDNA was then divided into two groups and ligated with two different adaptors. After the tester cDNA was hybridized twice with the driver cDNA and underwent two times of nested polymerase chain reaction （PCR）, the amplified cDNA fragments were subcloned into pGEM-Teasy plasmid vectors to construct the subtractive library. The library was amplified by E. coli strain DH5α. The randomly picked cDNA was sequenced and analyzed in the GenBank after the PCR. RESULTS： The cDNA subtractive library of HBV PTP1 transactivating genes was successfully constructed. The amplified library contained 60 positive clones, from which 32 inserts with 200-1 000 bp in length were obtained. Three differentially expressed protein genes and 4 sequences with unknown function were found by sequence analysis. CONCLUSION： The obtained genes may code the proteins involved in the regulation of cell cycle, metabolism, immunity and cell apoptosis.