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目的 应用抑制性消减杂交技术构建乙型肝炎病毒 (HBV)截短型中蛋白 (MHBst)反式激活基因差异表达的cDNA消减文库 ,克隆HBV截短型中蛋白反式激活相关基因。方法 以HBV截短型中蛋白表达质粒 pcDNA3.1( ) Mt16 7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HBV截短型中蛋白反式激活基因差异表达的cDNA消减文库。文库扩增后得到 94个白色克隆 ,进行菌落PCR分析 ,均得到 2 0 0～ 80 0bp插入片段。挑取 5 0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 2 3种编码基因 ,包括 19种已知基因和 4种未知基因。结论 筛选到的cDNA全长序列 ,包括一些与细胞生长调节、免疫应答及肿瘤发生密切相关的蛋白编码基因 ,因此可能是HBV截短型中蛋白反式激活靶基因
Objective To construct a subtractive cDNA library of hepatitis B virus (HBV) truncated protein (MHBst) transactivator gene by suppression subtractive hybridization and cloned HBV truncated transactivator - associated protein. Methods The HepG2 cells were transfected with pcDNA3.1 () Mt16 7, and the empty vector pcDNA3.1 () was used as a control. The transfected cell lysate was prepared and the mRNA was extracted and reverse transcribed into cDNA. After digestion with RsaI, the cDNAs were divided into two groups and ligated with two different linkers respectively. Two subtractive hybridization and two inhibitory polymerase chain reaction (PCR) / A vector to construct a cDNA subtractive library, which was then transfected into E. coli for library amplification. Randomly selected clones were amplified by PCR, sequenced and their homologies analyzed. Results The subtractive cDNA library of human truncated truncated protein transactivator gene was successfully constructed. Ninety-four white clones were obtained after amplification of the library, and colony PCR analysis was performed to obtain 20000 bp to 8080 bp inserts. Fifty inserts were sequenced and their full-length gene sequences were obtained by bioinformatics analysis. As a result, 23 coding genes were obtained, including 19 known genes and 4 unknown genes. CONCLUSION: The full-length cDNA sequence of the selected cDNAs, including some protein-coding genes closely related to cell growth regulation, immune response and tumorigenesis, may be the target gene of trans-activation of HBV truncated messenger protein