Abstract:

BACKGROUND: Bone morphogenetic protein (BMP) is a protein possesses potential activity, which can increase and enhance its activity when the bone issues are damaged, so it can be used to repair the bone defects when combined with carrier. However, there are few reports concerning it as gene therapy.
OBJECTIVE: To construct recombinant retroviral vector expressing human BMP2 gene and to discuss its biological function in osteoblasts.
METHODS: BMP2-specific primers were designed and synthesized according gene sequence of human BMP2 gene in Genbank，then BMP2 gene was amplified by Hifi PCR, which was recombined with cloning vector pDNR-CMV homologously into pDNR-CMV-BMP2 plasmid identified by BMP2 PCR and enzyme digestion of SalI and EcoRI as well as gene sequencing; recombinant plasmid pDNR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombined homologously in loxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67 and the supernatant was collected to assay viral titre. Human osteoblast was infected with retrovirus, then the growth of cells were observed by MTT, and the expression of BMP2 protein was detected by Western blotting at 48 hours transfection
RESULTS AND CONCLUSION: Digestion, BMP2 PCR and gene sequencing of pDNR-CMV-BMP2 were coincided with expected. After transfected with plasmid pLP-LNCX-BMP2, PT67 cells could be screened with G418 only to get stably integrated in BMP2, of whose supernanant viral titre amounted to 5×10⁸ pfu/mL. MTT assay showed that there had no evident difference in cellular inhibition between the normal and retrovirus groups at 72 hours after transfection (P > 0.05); Western blotting showed that the BMP2 was strong expressed at 48 hours after transfection. It demonstrated that BMP2 gene was successful cloned and its retrovirus vector was constructed.