靶向hVEGF165基因shRNA质粒表达载体的构建及筛选

Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting hVEGF_(165) gene

目的构建编码hVEGF165mRNA的shRNA质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体。方法以hVEGF165mRNA编码区中第5和第7外显子序列作为RNA干扰靶点,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体并通过PCR进行鉴定。经鉴定后分别转染稳定表达hVEGF165基因的BHK细胞,实时荧光定量PCR和Western blotting分别从mRNA和蛋白质水平检测抑制效果。结果构建的质粒表达载体PCR鉴定均可扩增出预期条带,构建成功。靶向hVEGF165基因的shRNA对所转染稳定表达hVEGF165基因的BHK细胞中hVEGF165基因mRNA和蛋白质表达均有抑制作用,其中shRNA2最为明显。结论成功构建了靶向hVEGF165基因的shRNA质粒表达载体,其中抑制效果最为明显的shRNA质粒表达载体为pDC316-EGFP-U6-shRNA2质粒。

Objective To construct a plasmid expression vector coding for the short hairpin RNA （shRNA） targeting hVEGF165 mRNA. Methods Three plasmid expression vectors coding for shRNA targeting exons 5 and 7 of hVEGF165 gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into BHK cells expressing hVEGF165 gene via ViofectTM reagent. The hVEGF165 gene silencing effect was detected by quantitative RT-PCR and Western blotting. Results The expected band was amplifiedfrom the plasmids coding for shRNA by PCR. Transfection of BHK cells expressing hVEGF165 gene with the shRNA plasmids resulted in a inhibition ofhVEGF165 mRNA and protein expressions by 77% and 70%, respectively. Conclusion The plasmid expression vectors coding for shRNA targeting hVEGF165 mRNA have been constructed successfully, of which pDC316- EGFP-U6-shRNA2 most effectively silences hVEGF165 gene in BHK cells.