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目的:构建用于RNAi的shRNA(small hairpinRNA)表达载体及检测其对低氧诱导因子-1(Hypoxia-inducibleFactor-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HIF1cDNA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。
OBJECTIVE: To construct shRNA expression vector for RNAi and to detect its silencing effects on Hypoxia-inducible Factor-1 (HIF1) gene. Methods: The H1 gene promoter was amplified by PCR from the human blood genome and cloned into the pEGFP-C1 vector fragment. The vector was named as pWH1. Primers were designed based on the human HIF1 cDNA gene, annealed and cloned into pWH1. The new vector was transfected into SGC7901 cells, then the expression of HIF1 gene was detected by RT-PCR and Western blot. Results: The pWH1 vector was able to express shRNA targeting HIF1 gene. The results of RT-PCR and Western blot showed that the expression of HIF1 mRNA and protein were significantly decreased. Conclusion: The shRNA expression vector pWH1 was successfully constructed, which is of great significance for the study of gene function.