shRNA表达载体pWH1的构建及用于HIF1基因的沉默

目的:构建用于RNAi的shRNA(small hairpin RNA)表达载体及检测其对低氧诱导因子-1(Hypoxia-inducible Factor-1,HIF1)基因的沉默效果.方法:从人血基因组中PCR扩增出H1基因启动子,克隆入酶切处理后的pEGFP-C1载体片段中,此载体命名为pWH1.以人HIF1 cDNA基因为靶标设计引物,退火后克隆入pWH1.新的载体转染SGC7901细胞,然后用RT-PCR和Western blot检测HIF1基因的表达改变.结果:构建的pWH1载体能很好地表达针对HIF1基因的shRNA,RT-PCR和Western blot的结果显示HIF1基因的mRNA和蛋白表达水平均明显下降.结论:成功构建了shRNA表达载体pWH1,这对于基因的功能研究具有重要的意义.

Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene

AIM:To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene. METHODS: The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot. RESULTS: The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramaticaly in mRNA and at protein level. CONCLUSION: The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.