抑制性消减杂交构建乙型肝炎病毒X蛋白羧基端缺失40个氨基酸cDNA文库

Construction of cDNA library of hepatitis B virus with X protein C-terminally truncated 40 amino acids by suppression subtractive hybridization method

目的：应用抑制性消减杂交（SSH）技术筛选乙型肝炎病毒（HBV）X蛋白羧基端缺失40个氨基酸（HBx3＇-40）反式激活基因,克隆其反式激活的相关靶基因.方法：以HBx3＇-40蛋白表达质粒pcDNA3（-）-HBx3＇-40 转染Huh-7细胞,以转染pcDNA3（-）-HBx为对照;制备转染后的细胞裂解液,提取总RNA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pUCm-T载体连接,构建cDNA消减文库,并转染大肠杆菌JM109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：成功构建了HBx3＇-40反式激活基因差异表达的cDNA文库,文库扩增后得到154个白色克隆,经菌落PCR分析,克隆包含有200～800 bp的插入片段,部分片段编码蛋白涉及原癌基因、信号转导相关基因、细胞生长因子相关基因、细胞凋亡、代谢和蛋白合成等相关基因.结论：应用抑制性消减杂交技术成功构建了HBx和HBx3＇-40差异表达的cDNA消减文库,为进一步阐明HBx3＇-40在HBV感染相关的肝细胞癌发生中的分子生物学机制提供理论依据.

Objective：To construct subtractive cDNA library from human hepatocellular carcinoma cells transactivated by C-terminally truncated 40 amino acids using suppression subtractive hybridization （SSH） technique and to clone the associated genes. Methods： Hub-7 cells were separately transfected with pcDNA3（-） harboring the sequence of HBx protein C-terminally truncated 40 amino acids and pcDNA3（-） harboring the full length sequence of HBx protein vectors. The total RNAs were isolated from the transfected Hub-7 cells and were reversely transcripted into double strand cDNAs. After the cDNAs were digested with restriction enzyme Rsa I , they were divided into 2 groups and were ligated to the special adaptor 1 and adaptor 2R, respectively. The tester cDNAs were then hybridized with driver cDNAs twice and the products were amplified twice by nested PCR technique. The PCR products were connected with pUCm-T plasmid vectors to establish the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The inserts of cDNAs were sequenced and analyzed in GenBank with Blast search. Results.. The subtractive cDNA library was successfully constructed. The amplified library contained 154 positive clones, and colony PCR showed that these clones contained 200-800 bp inserts： some fragments coded proteins involved proto-oncogenes, cell signaling genes, cell growth factor genes, cell apoptosis genes, metabolism and protein synthesis genes. Conclusion： Subtractive cDNA library has been successfully constructed by SSH technique, which may help to clone novel genes transactivated by HBx C-terminally truncated 40 amino acids and to explore the molecular mechanism of bepatoma patbogenesis.