Construction And Identification Of The Replication-Defective Recombinant Adenovirus Expressing Human Bone Morphogenetic Protein-4

Objective For further use in gene therapy of bone tissue engineering, we construct replication-defective recombinant adinovirus including the target gene fragment hBMP-4.Methods hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transfomed into packaging cell lines HEK293 to produce recombinant adenoviruses. These replication-defective recombinant adinovirus were transfected into HEK293 and HeLa cells. Then, total RNA and total protein was detected by RT-PCR and Western-blot.Results The pAdE/hBMP-4 was confirmed by restrictional endonuclease digestion; In HEK293 and HeLa cells, the specific transcription of hBMP-4 gene was confirmed by RT-PCR and the expression of hBMP-4 protein was confirmed by Western-blot assay.Conclusion The replication-defective recombinant adinovirus expression vector containing hBMP-4 gene has been constructed and expressed successfully. This has laid a foundation for the further research of genetherapy of hBMP-4.