【摘要】 目的构建携EGFP的人低氧诱导因子(HIF-1α)真核表达载体,为研究人HIF-1α基因对冠心病的血管新生作用奠定基础。方法采用分子克隆技术,KpnⅠ、XbaⅠ双酶切pcDNA3.1/V5-HisA-HIF-1α获得HIF-1αcDNA,克隆到质粒pcDNA3.1(+),得到质粒pcDNA3.1(+)-HIF-1α,以KpnⅠ,ApaⅠ双酶切质粒pcDNA3.1(+)-HIF-1α获得HIF-1αcDNA,克隆到质粒pEGFP-c1得到pEGFP-HIF-1α-c1质粒。脂质体法转染HEK293细胞,用荧光显微镜和Western blotting检测EGFP和HIF-1α在HEK293细胞的表达。均显示融合蛋白在细胞中表达。结果经酶切鉴定及PCR证实重组pEGFP-HIF-1α-c1质粒构建成功,荧光显微镜和Western blotting显示EGFP和HIF-1α融合蛋白在HEK293细胞中表达。结论成功构建重组真核表达载体pEGFP-HIF-1α-c1并在HEK293细胞表达,为冠心病的HIF-1基因治疗研究奠定更直观的基础。更多还原

【Abstract】 [Objective] To construct the eukaryotic expression vector containing the EGFP-HIF-1α gene for studying therapeutic angiogenesis of coronary heart disease. [Methods] Human HIF-1α cDNA obtained from the plasmid pcDNA3.1/V5-HisA-HIF-1α was cloned into plasmid pcDNA3.1 (+) with double digestion of Kpn1 and Xba1. HIF-1α cDNA obtained from pcDNA3.1 (+) -HIF-1α was cloned into plasmid pEGFP-c1 with double digestion of KpnⅠand ApaⅠ. The recombinant plasmid pEGFP-HIF-1α-c1 was transfected into the HEK293 cell by lipofectamine 2 000. The result was examined using fluorescent microscope. The expression of HIF-1α was delected by Western blotting. [Results] The recombinant pEGFP-HIF-1α-c1 was correctly constructed and confirmed by restriction endonuclease analysis and PCR amplification. The expression of EGFP can be seen by green-fluorescent microscope. The 120KDa of HIF-1α were delected in the HEK 293 cells by Western blotting. [Conclusion] The recombinant eukaryotic expression vector pEGFP-HIF-1α-c1 have been successfully constructed with efficient expressions in HEK293 cells.更多还原