Effect of recombinant adeno-associated BMP-4/7 fusion gene on the biology of BMSCs

Abstract

The aim of the study was to investigate the effect of recombinant adeno-associated virus (AAV) with morphogenetic protein 4/7 fusion gene on the ossification of rabbit bone marrow stromal cells (BMSCs). The genes BMP-4 and BMP-7 were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was generated through recombination. The rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with various multiplicity of infection (MOI) values. Expression of fusion gene BMP-4/7 protein was determined by the ELISA method. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. We successfully constructed the recombinant AAV with the BMP-4/7 fusion gene. A 29-20 kDa protein was shown by SDS-PAGE electrophoresis following transfection with AAV-BMP-4/7. The fusion protein BMP-4/7 in BMSCs transferred by AAV showed a positive correlation with various MOI values. There was significantly higher ALP and OC activity in the AAV-BMP-4/7 transfection groups than in the AAV-EGFP groups (t(ALP)=896.88, P<0.001, t(OC)=543.24, P<0.01). The fusion gene BMP-4/7 is capable of enhancing the expression of BMPs and possesses significant ossification activity through AAV.