Construction and expression of human hypoxia inducible factor-1α and enhanced green fluorescent protein eukaryotic expression vector

[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.