Objective     To construct adenovirus vector containing the enhanced green fluorescent protein (EGFP)-HIF-1 alpha gene for studying therapeutic angiogenesis of coronary heart disease.      Methods     Human HIF-1α cDNA obtained from the plasmid pcDNA3.1(+)-HIF-1 alpha with double digestion of KpnⅠ and ApaⅠ was cloned into plasmid pEGFP-C1. EGFP-HIF-1 alpha cDNA obtained from pEGFP-C1 with double digestion of NheⅠ and ApaⅠ was cloned into plasmid pShuttle2. The expression casse

tte containing EGFP-HIF-1 alpha cDNA was obtained from the recombinant pShuttle2-EGFP-HIF-1 alpha with double digestion of PI-Sce Ⅰ and I-Ceu Ⅰ

then ligated to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine2000. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and the titer was determined.      Results     The recombinant pAdeno-EGFP-HIF-1 alpha was correctly constructed and confirmed by restriction endonuclease analysis and PCR amplification. The transfected HEK293 cells were lysed by freeze-thawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 2×10

9

nfu/ml.      Conclusion     The recombinant adenovirus containing the EGFP-HIF-1 alpha gene was successfully constructed. It provides the further appreciable foundation of HIF-1 alpha gene therapy for coronary heart disease.