Construction of recombinant adeno-associated virus carrying human bone morphogenetic protein-7 and in vitro infection of bone marrow mesenchymal stem cells

Objective To construct the non-pathogenic recombinant adeno-associated virus(AAV) simultaneously carrying human bone morphogenetic protein-7 gene and green fluorescent protein(GFP) label,and assess its biological activity and function by infecting rabbit bone marrow mesenchymal stem cells(BMSCs) in vitro.Methods Plasmid pAAV-hBMP7-IRES-GFP was constructed by hBMP-7 segments amplified from plasmid pBluescript KS-hBMP7 carrying hBMP7 gene.The recombinant expression plasmid pAAV-hBMP7-IRES-GFP was co-transfected into AAV-293 cells with pAAV-Helper and pAAV-RC for recombinant AAV replication and package through homologous recombination.In vitro cultured rabbit BMSCs were infected with this recombinant virus.The virus titer was measured by infecting AAV-HT1080.Through infecting rabbit bone marrow mesenchymal stem cells in vitro,the optimal MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting.The BMP-7 gene expression was detected by Western blot assay,and its biological activity was assessed by osteogenic assay in vitro.Results The hBMP-7 gene was successfully amplified and recombinant pAAV-hBMP7-IRES-GFP was verified by double digestion.The purified recombinant virus had a high titer of 3.6×1011v.p./mL.The optimal MOI of BMSCs transfected with rAAV was 5×104 v.p./cell.Western blot showed that expression of the BMP7 genes in rAAV-hBMP7-IRES-GFP group was higher than that in the rAAV-IRES-GFP group.BMP proteins secreted from BMSC transfected with rAAV-hBMP7-IRES-GFP enhanced osteogenesis in vitro.Conclusion