Bone morphogenetic protein 2 plays a key role in inducing osteogenesis. It involves in a series of bioprocess, including cell proliferation, determining the differentiation direction of germ line and cell death.OBJECTIVE:
To construct and identify the recombinant adenovirus vectors encoding human bone morphogenetic protein 2 gene by using AdMax system.METHODS:
First, human bone morphogenetic protein 2 gene sequencing was amplified by PCR from human cDNA template and then cloned. Second, the recombinant shuttle plasmid was constructed and transformed into Escherichia coli competent cells DH5α. After the positive colonies were identified by colonies PCR and sequencing, the expression vectors were amplified and extracted. Next, the adenovirus expression vectors with target gene and the helper packaging plasmid carrying a majority of adenovirus genes were co-transfeced into 293 cells for virus packaging and amplification. Finally, target genes were detected by PCR, and target protein was detected by Western blot method, as well as infectious titer of the recombinant adenovirus was detected by end point dilution method.RESULTS AND CONCLUSION:
Gene fragment of a length of 1 223 bp human bone morphogenetic protein 2 was obtained by PCR. The expression vectors constructed by homologous recombination techniques were identified by PCR cloning and sequencing; the results were correct. After virus packaging and amplification in 293 cells were identified by Western blot and PCR methods, the virus titer of recombinant adenovirus was 5×1013 pfu/L. These results suggest that the recombinant adenovirus vectors carrying human bone morphogenetic protein 2 gene have been constructed successfully.