针对Cox-2基因siRNA载体构建和沉默效应  

Construction of a siRNA Vector Targeting Human Cox-2 Gene and Silence Effect

作　　者：赵国新[1] 徐慧丹[2] 赵新合 冯龙[4] 卫艳萍[4] 胡军[4] 

机构地区：[1]河南省医学科学研究所免疫室,河南郑州450052 [2]淮阳卫生学校,河南淮阳466700 [3]郑州市武警支队卫生队,河南郑州450052 [4]郑州大学基础医学院,河南郑州450052

出　　处：《河南科技大学学报（医学版）》2007年第1期1-4,共4页Journal of Henan University of Science & Technology:Medical Science

基　　金："十五""211工程"重点学科建设项目(教重办2002第2号)

摘　　要：目的构建针对人Cox-2基因的小干扰RNA(siRNA)表达载体,观察RNA干扰对肺癌细胞Cox-2基因表达的沉默作用。方法设计Cox-2靶向的发夹状siRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入pSINsi-hU6载体,转化扩增后进行序列测定。用脂质体包裹转染人肺癌细胞NCI-H446,采用RT-PCR检测Cox-2基因mRNA表达的变化。结果把针对Cox-2基因的siRNA的双链寡核苷酸片段克隆入pSINsi-hU6载体,经过酶切鉴定与测序,结果正确;RT-PCR检测显示,Cox-2基因的表达水平明显降低。结论成功构建出显著沉默细胞Cox-2基因表达的siRNA载体。Objective To construct a small interfering RNA （siRNA） expression vector targeting human Cox-2 gene and to observe the effects of gene silencing of Cox-2 by RNA interference on lung carcinoma cells. Methods Hairpin-shaped small interference RNA targeting Cox-2 gene was designed and the complementary single strands of DNA fragments were synthesized and annealed. The DNA fragments were recombinated into pSINsi-hU6 vector, followed by amplification and DNA sequencing, then transfected into human lung carcinoma cell line NCI-H446 mediated by Liposome. The change of the Cox-2 gene mRNA expression was detected by RT-PCR. Results The DNA fragments encoding Cox-2 targeted siRNA were cloned into the pSINsi-hU6 and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR revealed a strongly decreased expression level of Cox-2. Conclusion A siRNA vector which can silence Cox-2 gene expression effectively has been successfully constructed.

关 键 词：RNA干扰 COX-2 RT-PCR 

分 类 号：Q786[生物学—分子生物学]