靶向CGI-100基因shRNA干扰载体的构建与鉴定  

Construction and identification of the RNA interference system targeting CGI-100 gene

作　　者：汪永强[1] 张彦[1] 雷燕[1] 

机构地区：[1]重庆医科大学临床生化教研室重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2008年第12期1418-1422,共5页Journal of Chongqing Medical University

摘　　要：目的:构建靶向CGI-100基因的RNAi表达载体并鉴定其抑制效率。方法:设计及化学合成3对靶向CGI-100基因的短发夹结构寡核苷酸,经退火成双链DNA片段,将其与经限制性内切酶BamHⅠ和HindⅢ双酶切的PGenesil-1质粒连接,构建表达shRNA的重组质粒,用脂质体2000转染到K562细胞中进行表达,经RT-PCR和斑点杂交检测转染前后CGI-100基因表达的变化。结果:经DNA测序证实3对靶向CGI-100基因的RNA干扰表达载体PGenesil-1-CGI-100构建成功,其中第1对PGenesil-1-CGI-100在mRNA水平抑制白血病K562细胞中CGI-100基因表达的效率最高,达54%。结论:成功构建针对CGI-100基因的RNA干扰的表达载体,并筛选出1对抑制效率较高的重组干扰载体,为进一步研究K562细胞中CGI-100基因的功能奠定基础。Objective： To construct the expression vector of RNA interference（RNAi）system for CGI-100 gene and identify its inhibition efficiency. Methods： Three oligonucleotides targeting CGI-100 gene were devised and chemically synthesized,annealed to form double-strand DNA and inserted into plasmid PGenesil-1 whose segments were digested by BamH Ⅰ and HindlⅢ. The recombinant vector were then transfected into K562 cells with lipofectamineTM 2000.CGI-100 expresstion in the transfected ceils was assayed by RT-PCR and dot blot. Results： The recombinant plasmids of PGenesil-1-CGI-100 were successfully constructed by DNA sequencing analysis. The first of PGenesil-1-CGI-100 had the highest inhibition efficiency in mRNA levels,up to 54%. Conclusion： The RNA interference system targeting CGI-100 gene were successfully constructed and a interference vector with a higher inhibition efficiency was screened,which provides the foundation for further investigation.

关 键 词：CGI-100基因 RNA干扰 SHRNA K562细胞 

分 类 号：R733.7[医药卫生—肿瘤]