Construction and Identification of Recombinant Adenovirus Shuttle Plasimid pShuttle CMV-BMP2~+-IRES-GFP-1

Objective To clone bone morphogenetic protein 2(BMP2) gene into the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP-1 preparing for construction of 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 BMP2 gene contained in the plasimid of pcDNA3-BMP2 was sequenced, and the profile of restriction endonuclease sites existing in the sequence was analysed, then the base pairs behind the translation stop codon TAG were removed and a XhoⅠrestriction site was added following the 3’end of the mutant through polymeras chain reaction (PCR) mutagenesis technology. After being tested through sequencing, 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 the directional cloning method. To identify the correct recombinants, the analysis of restriction map was adopted. Results The restriction map of the recombinant cleaved by two kind of restriction endonucleases accorded with theoretic analyses of pShuttle CMV-BMP2~+-IRES-hrGFP-1. Conclusions The adenovirus shuttle plasmid pShuttle CMV-BMP2~+-IRES-hrGFP-1 has been constructed successfully.