【摘要】 目的:利用siRNA表达载体构建抑制人类心脏发育候选基因KLHL31表达的pSUPER RNAi载体(pSUPER-KLHL31)。方法:化学合成一对编码短发夹RNA序列的、靶向心脏发育候选基因KLHL31的寡核苷酸链60个碱基,退火,克隆到经BglⅡ、Xho Ⅰ双酶切的pSUPER质粒上,构建重组RNAi质粒(pSUPER-KLHL31),通过酶切鉴定及测序分析验证构建效果,将正确构建的质粒转染大鼠心肌细胞H9c2,建立产生逆转录病毒的细胞克隆。通过Western blotting检测转染pSUPER-KLHL31后细胞中KLHL31蛋白的表达量。结果:pSUPER-KLHL31载体经酶切鉴定及测序分析,结果表明60个碱基成功插入到预计位点,且序列完全一致;重组载体转染包装细胞,可表达绿色荧光蛋白,表明包装成功;Western blotting检测结果显示转染pSUPER-KLHL31的细胞中KLHL31蛋白表达量明显降低。结论:靶向KLHL31的pSUPER RNAi载体构建成功,为进一步从分子水平探讨KLHL31在心脏发育中的功能奠定了基础。更多还原

【Abstract】 Objective To construct a pSUPER RNAi vector that can inhibit human heart developmental candidate gene KLHL31 gene expression.Methods A pair of 60nt oligonucleotides coding for short hairpin RNA and targeting human heart developmental candidate gene KLHL31 were chemically synthesized and annealed and inserted into pSUPER plasmids digested with Bgl II and Xho I to construct the recombinant pSUPER RNAi plasmid(pSUPER-KLHL31).Recombinant pSUPER-KLHL31 plasmid was identified by enzyme digestion and sequencing analysis.The packaging cell H9c2 was transfected with the recombinant plasmid.Western blotting was used to determine the expression of the KLHL31 protein.Results The result of enzyme digestion and sequencing analysis demonstrated that 60 nt oligonucleotides had been inserted successfully into the vector.Green fluorescent was detected in virus-producing cells.Western blotting showed that pSUPER-KLHL31 can inhibit the expression of KLHL31 protein.Conclusion The pSUPER RNAi vector targeting human heart developmental candidate gene KLHL31 are successfully constructed and it can establish a foundation for our study the molecular function of KLHL31.更多还原