Construction and identification of human bone morphogenetic protein-7 recombinant adeno-associated virus type 2 vector and its expression in bone mesenchymal stem cells / 中华外科杂志

ABSTRACT

<p><b>OBJECTIVES</b>To facilitate 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.</p><p><b>METHODS</b>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 I 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 micro g/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 sulphate polyacrylamide gel electrophoresis. Following infection with rAAV2-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, MSCs were assessed qualitatively for BMP7 production.</p><p><b>RESULTS</b>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 Western-blot.</p><p><b>CONCLUSIONS</b>The hBMP7 recombinant adeno-associated virus vector is successfully constructed. The present in vitro study demonstrates that rAAV2-BMP7 could infect MSCs.</p>