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目的:应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)前-X基因反式激活基因差异表达的cDNA消减文库,克隆前-X反式激活相关基因,了解该段基因的可能生物学功能. 方法:构建表达质粒pcDNA3.1(-)-前-X,转染HepG2 细胞,以空载体pcDNA3.1(-)转染的HepG2细胞为对照;提取转染后细胞的mRNA,反转录为cDNA.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多聚酶链反应(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:成功构建前-X基因反式激活基因差异表达的cDNA 消减文库.文库扩增后得到45个白色克隆,进行茵落PCR 分析,均得到200-1000 bp插入片段.挑取含有插入片段的30个克隆进行测序,并通过生物信息学分析获得13种已知功能基因序列. 结论:应用SSH技术成功构建了前-X基因反式激活基因差异表达的cDNA消减文库.该文库的建立为阐明该基因生物学功能提供理论依据.
OBJECTIVE: To construct a subtractive cDNA library of differentially expressed transactivator genes of pre-x gene of hepatitis B virus (HBV) before cloning and analyze the possible gene of this gene by cloning the gene of -X transactivation Biological function.Methods: HepG2 cells were transfected with pcDNA3.1 (-) - pre-X, and HepG2 cells transfected with empty vector pcDNA3.1 (-) were used as controls. The mRNA, Reverse transcribed into cDNA.cDNA RsaI digestion, the experimental group cDNA was divided into two groups, respectively, with two different linkers convergence, and then with the control group cDNA two subtractive hybridization and twice inhibition of polymerase chain reaction (PCR) , And the product was ligated with pGEM-Teasy vector to construct a cDNA subtractive library, which was then transfected into Escherichia coli for library amplification, and randomly selected clones were amplified by PCR for sequencing and homology analysis.Results: Activation of gene subtractive cDNA subtractive library.After amplification of the library, 45 white clones were obtained and PCR-amplified by PCR to obtain 200-1000 bp inserts.The 30 clones containing insert were sequenced and sequenced by bioinformatics Learn 13 kinds of known functional gene sequences. Conclusion: SSH subtractive cDNA library was constructed successfully, which provided a theoretical basis for elucidating the biological function of this gene.