Gadd45a基因真核表达载体及其RNA干扰载体的构建  

Construction and Expression of the Gadd45a Gene and Its shRNA

作　　者：郭艳合[1] 刘立[1] 钱程[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2009年第4期578-582,共5页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：863项目(2006AA02Z126);973计划(2004CB518804);浙江省科技厅重点项目(2006C23006)

摘　　要：采用反转录PCR的方法,从人类肝组织细胞系L02中扩增Gadd45a基因的全长读码框,并将其克隆到pCDNA3.0上,该重组质粒命名为pC/Gadd45a。同时,采用化学方法合成Gadd45a基因的干扰序列,经退火后克隆至pCDNA3.0,命名为pC/shRNA。实验结果证实重组质粒pC/Gadd45a和pC/shRNA构建成功。然后,将pC/Gadd45a和pC/shRNA转染L02细胞后发现,Gadd45a基因在mRNA水平和蛋白水平上分别提高表达和降低表达。Gadd45a真核表达载体及其干扰载体的成功构建,为深入研究Gadd45a蛋白对肿瘤发生发展的作用以及该蛋白在细胞信号网络中的作用提供参考。To construct the specific eukaryotic expression vector of Gadd45α and its RNAi and induce their expression in the normal cell L02, which prepared for our further study. Gadd45α was amplified by reverse-transcription PCR from L02 cells and ligated with the vector pcDNA3.0, named as pC/Gadd45α. pC/shRNA was constructed by ligating the pcDNA3. 0 and the DNA fragment which annealed by two chemical synthesis ssDNAs. The recombinants and the pcDNA3.0 were transfected into L02 cells using LipofectamineTM 2000, and the expression of Gadd45α and its shRNA were detected by RT-PCR and western blotting. The data showed that eukaryotic vector pC/Gadd45α and pC/shRNA were constructed successfully. L02 cells transfected pC/Gadd45α and pC/shRNA could enhance and reduce the expression of Gadd45α, respectively.

关 键 词：GADD45A 癌症 PCDNA3.0 

分 类 号：Q786[生物学—分子生物学]