Construction And Functional Identification Of Human LMP-1 Gene Recombinant Adenovirus Vector

Background LIM mineralization protein-1 (LMP-1) is a novel intracellular osteogenic factor associated with bone development that has been implicated in the bone morphogenetic protein (BMP) pathway. Objective To construct the recombinant adenovirus vector containing human LIM mineralization protein-1 (LMP-1) gene by using the Ad-Easy system and study the mechanism of bone formation induced by LMP-1after infect dog bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Prepared pAdTrack-CMV and pUC57-LMP-1to generate a recombinant plasmid pAdTrack-CMV-LMP-1 after digested by the enzyme Kpnâ… and Hindâ…¢restriction enzyme digestion. Homologous recombination of pAdTrack-CMV-LMP-1 and adenovirus backbone plasmid pAdEasy-1 occurred in the BJ5183 after lineafized by PmeI to generate the recombinant plasmid pAd-LMP-1. Then packaged and amplified into replication-defective recombinant adenovirus Ad-LMP-1in the human embryonic kidney 293 cells (HEK293). Titers were determined after amplification and purification. Dog BMSCs were infected by Ad-LMP-1in the best value of MOI in vitro. The gene expression of LMP-1and BMP-7 was detected by RT-PCR and Western Blot. Treated dogs BMSCs with Ad-LMP-1 and rhBMP-7 to study the mechanism of bone formation of LMP-1 underwent mineralization (Ca) nodules 21d by alizarin red staining. Resultsâ‘ Ad-LMP-1 was successfully constructed and obtained titers in the packaging about 2.2x109efu/ml after amplified in HEK293.â‘¡The best efficiency can get at the most appropriate MOI 100 after Ad-LMP-1 infected BMSCs. Infected BMSCs can express mRNA and protein of LMP-1, but not caused expression of BMP-7.â‘¢Dog BMSCs were treated with Ad-LMP-1 and(or) rhBMP-7 for 21 days. We found that Ad-LMP-1 with exogenous rhBMP-7 promote transformation into osteoblasts together. Neither Ad-LMP-1 nor rhBMP-7 alone induced any transformation by day 21. Conclusion The recombinant adenoviral vector carring hLMP-1 gene is successfully constructed and express effectively in dogs BMSCs. Confirmed that the mechanism of bone formation induced by LMP-1 is synergistic effect with the performance of exogenous rhBMP-7.