siRNA靶向Rac1真核表达载体的构建与效应检测

目的 构建、纯化并鉴定Rac1特异性siRNA的真核表达载体,并在HeLa细胞中初步检测其转染率及对Rac1基因的抑制率.方法 体外合成含针对人、鼠的Rac1特异基因序列的siRNA链,定向克隆至pSUPER质粒中, 对重组质粒进行限制性内切酶酶切鉴定和测序分析.将抽提的质粒转染入HeLa细胞,荧光显微镜观察转染效率,荧光定量RT-PCR法鉴定其抑制Rac1基因表达的效果.结果 利用RNAi技术成功构建抑制 Rac1 表达的小干扰 RNA 重组载体,并成功转染到HeLa细胞中(载体转染率达到70%以上).在mRNA水平对Rac1基因的表达产生明显抑制作用(抑制率为87%以上).结论 成功构建了针对Rac1的siRNA真核表达载体,为进一步研究Rac1的功能奠定了基础.

Construction and Identification of Eukaryotic Expression Plasmid Expressing siRNA Targeting Rac1 and Detection of Its Specific Downregulation Effect

Objective To constructed eukaryotic expressing siRNA sections targeting human and rat Rad and to observe their effects on Rad gene expression in HeLa cell line. Methods siRNA sequences were designed and synthesized targeting human and rat's Rad mRNA, and then directionally cloned into pSUPER plasmid. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into HeLa cell line and the transfection rate was evaluated with fluorescence microscope. The effects of the recombinants on Rad at mRNA levels were observed by RT-PCR. Results Three siRNA expressing recombinants and corresponding negative control vector were constructed and transfected into HeLa cell successfully. Rad transcript was reduced significantly in three transfectants. Conclusion The construction of eukaryotic expression vectors expressing siRNA sections targeting Rad and identification successfully. This will be very helpful for further study on the function of Rad and it may be a useful tool in the treatment of the neovascular retinopathy.