血红素氧合酶-2基因真核表达载体的构建

Construction of HO-2 Gene Eukaryotic Expression Vector

为构建血红素氧合酶-2(HO-2)真核表达载体pEF1α-HO-2-AcGFP,并观察其在小鼠脑血管内皮细胞中的表达情况。试验利用PCR技术从C57BL/6小鼠海马组织中扩增出HO-2基因cDNA序列,用双酶切法将此序列克隆到真核表达载体pEF1α-IRES-AcGFP上,构建HO-2的真核表达载体pEF1α-HO-2-AcGFP,重组载体经EcoRI和BamHI双酶切和测序鉴定。电穿孔法转染小鼠脑血管内皮细胞,48h后,实时定量PCR、Western blot检测HO-2在小鼠脑血管内皮细胞中mRNA和蛋白质水平的表达情况。结果表明,pEF1α-HO-2-AcGFP载体构建正确;重组载体转染小鼠脑血管内皮细胞后HO-2在mRNA水平表达量较空载体转染极显著增高(P<0.01),在蛋白水平表达量较空载体转染显著增高(P<0.05)。表明HO-2基因的真核表达载体pEF1α-HO-2-AcGFP构建成功,为进一步研究其机制和功能奠定了基础。

To construct the heme oxygenase-2 eukaryotic expression vector, and observe the expression of cerebrovascular endothelial cells in mice, the PCR technology was used to amplify the HO-2 gene cDNA sequence from C57BL/6 mice hippocampal tissue, which was cloned into eukaryotic expression vector pEFlα-IRES-AcGFP by double enzyme digestion method. Recombinant vector pEFlα-HO-2-AcGFP was confirmed by EcoR I and BamH I double enzyme and sequencing. Then cerebrovascular endothelial cells were transfected with the recombinant eukaryotic expression vector by electroporation technology. The Real-time PCR and Western blot was used to detect HO-2 the expression of mRNA and protein levels in cerebrovascular endothelial cells. The eukaryotic expression vector pEFlα-HO-2-AcGFP was corrected by double enzyme digestion and DNA sequencing. The results showed that mRNA of HO-2 expression is sig- nificantly higher in cerebrovascular endothelial cells of recombinant vector group than of empty vector group （P〈0.01）, and protein levels is significantly higher （P〈0.05）. The eukaryotic expression vector pEFlα-HO-2-AcGFP was successfully constructed, which will provide a reference for the further study on the mechanisms and function of HO2 gene.