shRNA表达载体pWH1构建及用于HIF1基因沉默.docVIP

shRNA表达载体pWH1构建及用于HIF1基因沉默 　 作者：吴元明, 张晓楠, 韩者艺, 李春英, 陈南春 【摘要】 　　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。 【关键词】 shRNA 表达载体 RNA干涉 HIF1基因 　　[Abstract]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. 　　[Keywords]shRNA; expression vector; RNA interference; HIF1 gene 　　RNA干涉(RNA interference, RNAi)是将双链RNA（dsRNA）导入细胞引起特异基因mRNA降解的一种细胞反应过程。它是转录后基因沉寂（PTGS）的一种。1998年, Fire等[1]在利用反义核酸技术来抑制线虫基因表达时意外地发现, 由正义和反义RNA退火形成的dsRNA引起的基因表达抑制要比单独应用正义或反义RNA强10倍以上。dsRNA引起的基因表达抑制不是正义或反义RNA引起的基因表达抑制在数学上的叠加, 这就说明dsRNA触发了细胞内的一些反应机制, 从而引起了高效和特异的基因表达抑制效果。当时, 他们就把这种现象命名为RNAi。RNAi的应用谱非常广泛, 从单细胞生物一直到人。其中研究最多的就是线虫, 其次就是果蝇。哺乳动物细胞的RNAi应用研究并不象无脊椎动物那样进展得那么顺利。Wianny等[2]将dsRNA用显微注射的方法在小鼠胚胎成功进行了RNAi。Sayda等[3]用人工合成的21ntRNA二合体在培养的哺乳动物细胞成功进行了RNAi。Brummelka