[Construction and identification of human bone morphogenetic protein-7 recombinant adeno-associated virus type 2 vector and its expression in bone mesenchymal stem cells].

Objectives To facihtate gene therapy research using recombinant adeno-associated virus type 2 (rAAV2) vector as gene transfer vehicle,and to construct a rAAV2 based vector carrying bone morphogenetic protein-7 (BMP7)and observe its expression in bone mesenchymal stem cells.Methods The coding sequence (1.3 kb) of BMP7 was amplified by polymerase chain reaction (PCR) from the pcDNA1.1 (+) plasmid containing the human BMP-7 cDNA.After purified,the gene fragment was cloned into a plasmid pUC18 and termed plasmid pUC18-hBMP7.The recombinant pUC18-hBMP7 was digested by Kpn/and Sal I and further ligated to the pSNAV by T4DNA ligase.The resultant plasmid PSNAV-hBMP7 was transformed into DH5a Escherichia coli,and positive colonies were screened by PCR and digest with restriction enzyme to identify the correct recombinant clones.BHK-21 cells were transfected with the purified pSNAV-BMP7 plasmid according to a standard calcium phosphate precipitation method.The cells were then cultured in selection media containing 800 μg/ml G418 (Gibco/BRL).G418-resistant BHK-21 cell clones were isolated and the integrity of hBMP7 gene was determined by PCR using the above PCR primers.To package the virus,stably transfected BHK-21 cells were subsequently infected with recombinant herpes simplex virus type 1 (rHSV-1).The collected cells were processed by chloroform treatment,PEG8000/NaCl precipitation and chloroform extraction for purification.The titer was determined using quantitative DNA dot blots and the purity was examined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis.Following infection with rAAV2-BMP7 at multiplicities of infection of 1×10~5 vector genomes per cell and subsequent culture,MSCs were assessed qualitatively for BMP7 production.Results Transient transfection showed an efficiency of 98.8% in MSCs.RT-PCR showed that MSCs had transcription of BMP7 that was enhanced by the gene transfer.BMP-7 expression in MSCs was identified by Westeru-blot.Conclusions The hBMP7 recombinant adeno-associated virus vector is successfully constructed.The present in vitro study demonstrates that rAAV2-BMP7 could infect MSCs.