Study On Gene Therapy Of Myocardial Ischemia By RAAV8-Mediated HIF-1α Gene

Objective:To construct the plasmid vector of recombinant adeno-associated virus contains MHC myocardial specific troponin promoter and hypoxia induced factor-la gene(pAAV-MHC-TnT-HIF1α), and pack 6 type and 8 type recombinant adeno-associated virus, transduce the original generated myocardial cells In vitro, observe the gene expression in myocardial cells.Methods:1 Plasmid construct of pAAV-MHC-TnT-HIF1a.With existing plasmid pEMBL-CMV-GFP as templates, synthesize GFP gene fragment with PCR. Enzyme cut GFP fragment and existing plasmid pAAV-MHC-TnT-LacZ,construct a plasmid pAAV-MHC-TnT-GFP,Then enzyme cut plasmid pAAV-MHC-TnT-GFP and plasmid pGEM-HIF1a that contains HIF1a gene,construct a recombinant plasmid pAAV-MHC-TnT-HIF1a.2 Detect gene expression activity of TnT promoter and HIF-1a geneTransfect 293 cells with pAAV-MHC-TnT-GFP, observe GFP expression with fluorescence microscope after 48h. Transfect 293 cells with pAAV-MHC-TnT-HIF-1α, detect HIF-1a expression with Western Blot after 48h. 3 The virus package of rAAV-MHC-TnT-HIF-1 aTransfect 293 cells with pAAV-MHC-TnT-HIF-1 a, pHelper and pAAV8 or pAAV6. After virus purification, detect virus titer with Dot-blot, detect virus purity with SDS-PAGE.4 Detect the expression of rAAV-MHC-TnT-HIF-1αInfect original generated myocardial cell with rAAV6-MHC-TnT-HIF-1α, collect cell after 48h, detect the expression of HIF-1a with Western Blot.Results:1 Successful construct the packaging plasmid of pAAV-MHC-TnT-HIF 1 a.2 The expression of pAAV-MHC-TnT-GFP and pAAV-MHC-TnT-HIF1a is normal in 293 cells.3 Successful packaging two viruses of rAAV8-MHC-TnT-HIF1a and rAAV6-MHC-TnT-HIF1a,and virus titer no less than 1012/ml.4 rAAV6-MHC-TnT-HIF1a can express in original generated myocardial cell.conclusion:1 Successful construct a packaging plasmid of recombinant adeno-associated virus contains MHC myocardial specific troponin promoter and hypoxia induced factor-1a gene(pAAV-MHC-TnT-HIF-1a).2 Successful pack two recombinant adeno-associated viruses of rAAV8-MHC-TnT-HIF-1a and rAAV6-MHC-TnT-HIF-la, rAAV6-MHC-TnT- HIF-1a can normal express in original generated myocardial cells in vitro, rAAV8-MHC-TnT-HIF-1a can low express in 293 cell.And lay a solid foundation for further myocardial ischemia gene therapy in vivo.