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 × 10¹¹ v. 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: Recombinant rAAV-hBMP7-IRES-hrGFP was successfully constructed with a high virus titer and good infectibility, which may provide the basis for in vitro and in vivo experiments on gene therapy of bone regeneration.