Construction and identification of human hypoxia inducible factor-1αadenovirus vector

【Objective】To construct adenovirus vector containing the HIF1αgene for studying therapeutic angio-genesis of coronary heart disease. 【Methods】 Human HIF1αcDNA obtained from the plasmid pcDNA3.1/V5-HisA- HIF1αwas cloned into plasmid pcDNA3.1(+) and further cloned into plasmid pShuttle2. The expression cas-sette containing HIF1αcDNA was obtained from the recombinant pShuttle2 with double digestion of PI- Sce I andI- Ceu I, then ligated to Adeno- X Viral DNA with in vitro ligation. The recombinant adenoviral plasmid was identi-fied and transfected into the adenoviral packaging cell HEK293 by lipofectamine 2000 mediated gene transfermethod to pack the virus. The recombinant adenovius was confirmed by polymerase chain reaction (PCR) and thetiter was determined. 【Results】The recombinant pAdeno- HIF1αwas correctly constructed and confirmed by re-striction 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 ofrecombinant adenovirus. The viral titer was 3×109 pfu/mL. 【Conclusion】The recombinant adenovirus containing theHIF1αgene was successfully constructed. It provided the further foundation of HIF1αgene therapy for coronaryheart disease.