Construction Of Reconbinant Adenovirus Containing Human Mineralization Protein-1 And Expression Of Infected Bone Marrow Mesenchymal Stem Cells

Objective: Choose the AdEasy Adenovirus Vector System to conduct the recombinant adenovirus containing hLMP-1 gene, and detect the expression of hLMP-1 gene in infected rabbit BMSCs, to Lay the foundation for further animal experiment.Methods: hLMP-1 gene with a sequence encoding His-tag was amplificated by using pIRES2-EGFP-LMP-1 plasmid as a template for PCR with a specially designed downstream primer. Then the target gene was cloned to the pMD18-T vector for blue and white screening and sequencing. Once verified, the gene was cut out by double endonucleases, connected to the shuttle vector pAdTrack-CMV.The newly constructed vector was linearized by PmeI following efficient homologous recombination with the backbone vector pAdEasy-1 in BJ5183.The correct recombinant pAd-LMP-1 was selected by ampicillin resistance and endonucleases.Linearize pAd-LMP-1 with Pac I and transfect to HEK293 cell by means of mediated Lipofectamine.Then it was amplificated several times by infecting HEK293 cells,The titer of virus was measured after purification.BMSCs were isolated and culture-expanded from rabbit bone marrow.The third generation BMSCs were infected by recombinant adenovirus at different MOI,and then observed the efficiency of infection and the growth of cells.The mRNA and protein expression of LMP-1 was detected in BMSCs, which were infected wity Ad-LMP-1 at the most appropriate MOI, were detected by RT-PCR and Western blot respectively.Results: Digestion and sequencing analysis confirmed that the recombinant plasmid carrying hLMP-1 gene with His-tag was successfully constructed . Bone marrow stromal cells were isolated and cultured in vitro by a mature easy way. The best efficiency infection was 50%～70% ,which was get after the recombinant adenovirus infected BMSCs for 3 days.The effective expression of LMP-1 in infected cells has been proved by RT-PCR and Western Blot.Conlusion: The recombinant adenovirus containing hLMP-1 gene with His-tag was successfully constructed. The BMSCs infected with recombinant adenovirus Ad-LMP-1 can effectively express hLMP-1.