针对IER3IP1基因的shRNA真核表达载体的构建和鉴定  

Construction and identification of eukaryotic expression vector of short hairpin RNA for IER3IP1 gene

作　　者：雷燕[1] 张彦[1] 汪永强[1] 

机构地区：[1]重庆医科大学检验系临床生化教研室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》2009年第5期578-582,共5页Journal of Chongqing Medical University

摘　　要：目的:构建特异性针对IER3IP1基因的shRNA(Short Hairpin RNA)的真核表达载体,观察对IER3IP1基因表达的沉默作用,筛选出抑制效果较好的干扰片段。方法:针对IER3IP1基因,设计合成2对特异性DNA片段以及1对无关序列的阴性对照DNA片断,将它们插入真核表达载体pGenesil-1中构建重组质粒,质粒转染L02正常肝脏细胞株后,以半定量RT-PCR法检测转染后24、48、72hIER3IP1基因的抑制效果。结果:成功构建2个特异性针对IER3IP1的shRNA真核表达载体;所构建的载体中有1对能够特异性的降低L02细胞中IER3IP1的mRNA表达水平,且干扰效果显著,抑制率较高,与空载对照组比在转染后24、48、72h抑制率分别为64%、79%、69%。结论:设计构建的真核表达载体可以特异性干扰IER3IP1基因的表达,为进一步研究IER3IP1基因的功能奠定了基础。Objective： To construct the shRNA （Short Hairpin RNA ） eukaryotic expression vector targeting at IER3IP1 gene and observe its silencing effect on IER3IP1 gene. Methods： Two pairs of DNA fragments targeting at IER3IP1 gene and one pair of DNA fragment of negative control were synthesized and cloned into the Eukaryotic Expression Vector pGenesil-1.These vectors were separately transfected into the L02 cells. Inhibitory effect at the time of 24,48,72 h after transfection were detected by semiquantitative RT-PCR. Results： These shRNA eukaryotic expression vectors were successfully constructed. One of them could significantly and specially inhibite the expression of IER3IP1 gene and the inhibition ratios were separately 64% ,79% and 69% at the time of 24,48 and 72 h,compared with empty vector group. Conclusion： The combinated eukaryotic expression vector could inhibit the expression of IER3IP1 gene, which forms the basis for investigating IER3IP1 gene＇s function.

关 键 词：RNA干扰 基因 IER3IP1 

分 类 号：R733.7[医药卫生—肿瘤]