人血管内皮生长因子mRNA靶向siRNA表达载体的构建和表达

Vector construction and silencing effect of VEGF gene targeted small interfering RNA

目的:构建人血管内皮生长因子(VEGF)mRNA靶向siRNA表达载体,观察其表达情况。方法:设计人VEGF靶向的发夹样siRNA,依据设计,合成2条互补的寡核苷酸链,退火后连接入pSUPERneo-GFP载体,转化扩增后得到重组载体pSUPERneo-GFP-siVEGF,进行序列测定。用脂质体转染法将重组载体转染食管癌细胞株Eca109,采用RT-PCR检测转染细胞VEGF mRNA的表达水平。结果:经过酶切鉴定与测序,pSUPERneo-GFP-siVEGF构建成功。稳定转染该重组体的Eca109细胞VEGF mRNA表达水平明显降低。结论:成功构建了针对VEGF mRNA的siRNA载体,转染细胞后可显著抑制VEGF mRNA的表达。

Aim： To construct VEGF gene-targeted small interfering RNA（siRNA） vector. Methods： VEGF gene-targeted hairpin siRNA was designed, then two complementary oligo nueleotide strand were synthesized and inserted into pSUPERneo-GFP vector after annealing to get the new vector pSUPERneo-GFP-siVEGF, which was digested by restrictive enzyme and sequenced. After that, the new vector was transfeeted into human esophageal carcinoma Eea109 cell using lipofeetamine method, and the mRNA exprssion level of VEGF gene in cells was detected by RT-PCR. Results and Conclusion： pSUPERneo-GFP-siVEGF is built successfully,and the expression level of VEGF mRNA in transfected Eca109 was decreased.