Abstract:

BACKGROUND: The upstream and downstream genes can be co-transcribed by internal ribosome entry site (IRES) sequence, by which human bone morphogenetic protein 2 (hBMP2) and human fibroblast growth factor 2 (hFGF2) genes can be highly co-expressed, thus, it provides a new way for repairing bone defects.
OBJECTIVE: To construct the recombinant adenovirus vector co-expressing hBMP2 and hFGF2 depending on IRES sequence by λ phage-site specific recombination systems and to verify the correct recombination.
METHODS: BMP-2cDNA and FGF-2 cDNA were amplified by PCR, and were subcloned into the pIRES2-EGFP plasmid to obtain phBMP2-IRES-hFGF2 plasmid. It was subcloned into entry vector pDONR221 by BP reaction and subcloned into pAd/CMV/V5-DEST vector by LR reaction and linearization transfection 293 cells to obtain pAd-hBMP2-IRES-hFGF2.
RESULTS AND CONCLUSION: The target gene of hBMP2-IRES-hFGF2 was transferred into AD/CMV/V5-DEST vector correctly and it was confirmed by sequencing. The expression clone pAd-hBMP2-IRES-hFGF2 was packaged into maturated adenovirus successfully; the titer of Ad-hBMP2-IRES-hFGF2 was 2.82×1010 ifu/mL. The results demonstrated that adenovirus vector co-expressing hBMP2 and hFGF2 are constructed successfully.