Construction and Identification of a Novel Expressing BMP2 Recombinant Adenovirus Vector

Objective To construct a novel recombinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein(GFP) as a reporter on the same transcript.Methods The basic pairs of BMP2 behind the translation stopping codon TAG were removed and a XhoⅠrestriction site was added following the end of the mutant.Then,the mutant of BMP2 gene (BMP2 + gene) was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV - IRES - hrGFP - 1 by means of the directional cloning technology.Once tested by restriction digestion,the shuttle plasmid was linearized with Pme I and transformed into BJ5183 - AD - 1 cells to recombine the genes of BMP2 + and GFP together with their cis - acting elements into adenoviral plasmid through a new simplified bacterial homologous recombinant method.To expose its inversted terminal repeats,the recombinant adenoviral plasmids were digested with PacⅠ,and were then used to 293 transfected cells to generate the desired recombinant adenoviruses, Ad CMV - BMP2 + - IRES - GFP - 1.After the resultant viruses infected the bone marrow stromal cells(MSCs),the expression of BMP - 2 gene was verified by RT - PCR,the osteoinduction activity of the expressed BMP2 was confirmed by alkaline phosphatase activity assay,and the expression of GFP gene was tested through fluorescence microscope.As a positive contrast,another recombinant adenovirus,AdCMV -BMP2,which has been used extensively since many years ago,was also constructed.Results Either the sequence of BMP2 gene mutant or the restriction maps of the recombinant adenoviral shuttle plasmids and the recombinant adenoviral plasmids accorded with experimental project beforehand.The new recombinant adenovirus vectors could express both the gene of BMP2 and GFP at the same time.The expressed BMP2 had osteoinduction activity.Conclusions A novel recombinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein as a reporter on the same transcript were successfully constructed.