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目的:筛选与克隆TAHCCP2的反式激活基因,了解其可能存在的调节功能线索.方法:应用抑制性消减杂交(SSH)技术及生物信息学(bioin-formatics)技术筛选并克隆TAHCCP2反式激活的新型靶基因.以TAHCCP2表达质粒pcDNA3.1(-)-TAHCCP2转染HepG2细胞.以空载体pcDNA3.1(-)为平行对照,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TAHCCP2反式激活基凶差异表达的cDNA消减文库.文库扩增后得到70个阳性克隆,进行菌落PCR分析,均得到200-1000bp插入片段.对插入片段测序,并通过生物信息学分析获得其全长基因序列.结果共获得15种编码基因.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TAHCCP2可能存在的调控机制的线索,尚需进一步的实验证明.
OBJECTIVE: To screen and clone the transactivation gene of TAHCCP2 and to find out the possible regulatory clues of TAHCCP2.Methods: The transactivation of TAHCCP2 was screened and cloned by using SSH and bioinformatics techniques New target gene was transfected into HepG2 cells with TAHCCP2 expression plasmid pcDNA3.1 (-) - TAHCCP2.The empty vector pcDNA3.1 (-) was used as a parallel control to prepare the cell lysate after transfection, mRNA was extracted and reverse transcribed into cDNA , After 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 two inhibitory polymerase chain reaction (PCR) T / A vector to construct a cDNA subtractive library, and then transfected into E. coli to amplify the library.After randomly selected clones were amplified by PCR, sequencing and homology analysis were performed.Results: The successful expression of human TAHCCP2 transactivator cDNA subtractive library.After amplification by PCR, 70 positive clones were obtained and colony PCR analysis was carried out to obtain 200-1000bp insert.Sequencing the inserted fragment and obtaining the full-length gene sequence by bioinformatics analysis.Results A total of 15 Coding gene CONCLUSION: The full-length cDNA sequence screened out, including some protein coding genes closely related to cell growth regulation, material metabolism, immunity and apoptosis, may be clues to the possible regulatory mechanisms of TAHCCP2, which needs further experimental evidence.