构建shRNA真核表达载体抑制人COX-2的表达

目的构建编码人环氧合酶-2(COX-2)mRNA的shRNA真核表达载体质粒,特异性抑制人COX-2的表达。方法以人COX-2mRNA编码区中2条不同序列作为RNA干扰靶点,分别构建2个shRNA真核表达载体质粒,WBH1和WBH2,进行酶切鉴定和测序。用脂质体转染人胃癌细胞系SGC-7901,RT-PCR和Western blot分别从mRNA和蛋白质水平检测抑制效果。结果WBH-1高效特异地抑制了人COX-2的表达,抑制率达70.42%;而WBH-2未产生抑制效果。结论通过构建COX-2的shRNA真核表达载体导入细胞可以有效地抑制人胃癌细胞中COX-2的表达,其抑制效果与靶点的选择高度相关。

Silencing Human COX-2 Gene by Construction of Eukaryotic Expression Vector Expressing the Short Hairpin RNA

ObjectiveTo specific suppress the expression of human cyclooxygenase-2 (COX-2)gene by constructing the eukaryotic expression plasmid of shRNA.To study whether the technology of RNA interference could be a way of anti-cancer gene therapy.MethodsPlasmids containing two different sequences of human COX-2 mRNA coding region were constructed.We gained two plasmids WBH 1 and WBH 2.Plasmids were identified by enzyme cutting and sequencing.Detect the effect of inhibition by RT-PCR and Western blot after transfecting human gastric cancer cell line SGC-7901 with liposomes.ResultsThe expression of human COX-2 gene was suppressed specific and effectively by WBH 1.The efficiency of inhibition is 70.42%.Yet WBH 2 is not the same.ConclusionCOX-2 expression in human gastric cancer cells can be inhibited significantly by construction of eukaryotic expression vector expressing the shRNA.The inhibitory effect is highly related to selection of target sequence.