Construction and identification of human mutant hypoxia inducible factor-1α adenovirus vector

AIM: To construct adenovirus vector containing the mutant HIF1α gene and to study the effect of mutant hypoxia inducible factor-1α on the angiogenesis of coronary heart disease. METHODS: Human mutant HIF1α cDNA obtained from the plasmid pcDNA3.1(+)-HIF1α was cloned into plasmid pShuttle2. The expression cassette containing mutant HIF1α cDNA was obtained from the recombinant pShuttle2 with double digestion of PI-Sce I and I-Ceu I and then ligated to Adeno-X Viral DNA with in vitro ligation. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine2000 mediated gene transfer method to pack the virus. The recombinant adenovius was confirmed by polymerase chain reaction (PCR) and the titer was determined. RESULTS: The recombinant pAdeno-HIF1α was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. 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~(12) pfu/L. CONCLUSION: The recombinant adenovirus containing the mutant HIF1α gene has been successfully constructed, which paves the way for mutant HIF1α gene therapy of coronary heart disease.