Abstract:

BACKGROUND: The commonly used genetic carrier has a certain shortcoming, such as cannot be used directly in vivo, and using recombinant adeno-associated virus (AAV) as a vector carrying transforming growth factor-β1 (TGF-β1) to improve cartilage renovation has not been reported.
OBJECTIVE: To construct recombinant adeno-associated virus expressing TGF-β1, and assess the virus titer, and to identify the effect of recombinant adeno-associated virus on the infection activity of bone marrow mesenchymal stem cells.
METHODS: The TGF-β1 gene was amplified by PCR and directly cloned into the plasmid pAAV-TGF-β1-hrgreen fluorescent protein. The recombinant pAAV-TGF-β1-GFP was co-transfected into AAV-293 cells with pAAV-RC and pAAV-Helper for AAV-2 replication and packaging through homologous recombination. The virus titer was measured through infecting AAV-HT1080, and the recombinant virus was verified by PCR of the exogenous interest gene. Through infecting rabbit bone marrow mesenchymal stem cells in vitro, the infection efficiency and activity was detected.
RESULTS AND CONCLUSION: The TGF-β1 gene was successfully amplified and recombinant pAAV- TGF-β1- IRES-GFP was verified by double digestion and there was a 1.3 Kb target strap in accordance with TGF-β1. The system provided a high packing ratio and the purified recombinant virus has a high titer of 5.2×1011 v.g/mL. The recombinant virus was confirmed by exogenous human VEGF165 gene PCR. The rAAV-TGF-β1 -GFP could infect rabbit bone marrow mesenchymal stem cells at a ratio of 42% and express green fluorescence under fluorescent microscope. The rAAV-TGF-β1-GFP was successfully constructed with a satisfying biological ability of infecting bone marrow mesenchymal stem cells, which may offer the basement of gene therapy for cartilage repairation.