Hif-1α RNA干扰表达载体的构建及鉴定

Construction and identification of hif-1α shRNA recombinant plasmids

目的:构建hif-1α基因的RNA干扰表达载体pSilencer3.1-hif-1α,并检测其干扰Hela299细胞hif-1α表达的效率。方法:根据GenBank中hif-1α的mRNA序列,选择设计3对干扰序列,构建sihif-1α重组表达载体,测序正确后经脂质体转染Hela299细胞,Trizol试剂盒一步法提取细胞总RNA,应用RT-PCR检测hif-1α的mRNA的表达;单去污剂裂解法提取细胞总蛋白,Western blotting法检测蛋白表达。结果:测序结果显示,测定序列与设计序列完全相同,表明质粒构建正确。转染3个重组pSilencer3.1-sihif-1α表达载体后24h,Hela299细胞hif-1α的mRNA水平明显降低,对照组hif-1α/GAPDH比值为0.55,转染pSilencer-sihif-1α-1组hif-1α/GAPDH比值为0.13,两组比值比较差异有显著性(P<0.05),转染pSilencer-sihif-1α-2组hif-1α/GAPDH比值为0.33,两组比值比较差异有显著性(P<0.05),转染pSilencer-sihif-1α-3组Hif-1α/GAPDH比值为0.08,两组比值比较差异有显著性(P<0.01);转染3个重组pSilencer3.1-si Hif-1α表达载体后48h,HIF-1α蛋白的表达水平明显降低。结论:成功构建了Hif-1α基因的si RNA真核表达载体,该载体可有效抑制Hela299细胞hif-1α的表达。

Objective To construct the expression vectors of pSilencer3.1 -hif-1α and identify the inhibition of hif-1α in Hela299 cells after transfection with the combinative plasmids. Methods The interfering sequences of hif-1α were designed according to the sequence of hif-1α of GenBank. constructed by DNA ligase. Trizol reagent was used to Three recombinant plasmids of pSileneer3.1-hlf-1α were extract the whole RNA of cells and RT-PCR assay was applied to detect the expression of hif-1α mRNA . Lysis assay was used to extract the protein from cells and Western blotting was adopted to observe the expression of HIF-1α protein. Results The vectors were identified to be right after sequencing. The mRNA level was decreased 24 h after transfection with three vectors of pSilencer-hif- 1α, and the ratios of hif-1α /GAPDH in control, group transfecting with pSilencer-sihif-1α-1, group transfecting with pSilencer-sihif-1α-2, group transfecting with pSilencer-sihif-1α-3 were 0.55, 0.13, 0.33, and 0.08, respectively （P〈0.05, P〈0.05, P〈0.01）. The protein expression levels of HIF-1α 48 h after transfection with three vectors of pSilencer-hif-1α were decreased significantly. Conclusion Effective pSilencer-hif-1α plasmids are constructed successfully and they could interfere with the expression of hif-1α in Hela299 cells.