Construction and identification of human mutant hypoxia inducible factorª²1¦Á adenovirus vector

    GUO Shouª²Gui, WU Pingª²Sheng£¬WANG Yueª²Gang£¬FU Ruiª²Bin

    Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou 510515£¬ China

    ¡¾Abstract¡¿ AIM: To construct adenovirus vector containing the mutant HIF1¦Á gene and to study the effect of mutant hypoxia inducible factorª²1¦Á on the angiogenesis of coronary heart disease. METHODS: Human mutant HIF1¦Á cDNA obtained from the plasmid pcDNA3.1(+)ª²HIF1¦Á was cloned into plasmid pShuttle2. The expression cassette containing mutant HIF1¦Á cDNA was obtained from the recombinant pShuttle2 with double digestion of PIª²Sce I and Iª²Ceu I and then ligated to Adenoª²X 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 pAdenoª²HIF1¦Á was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK293 cells were lysed by freezeª²thawing 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¡Á1012 pfu/L. CONCLUSION: The recombinant adenovirus containing the mutant HIF1¦Á gene has been successfully constructed, which paves the way for mutant HIF1¦Á gene therapy of coronary heart disease. ......