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Published on: May 6, 2015
[Construction and identification of replication-defective recombinant adinovirus expressing human bone morphogenetic
Xingyu Wang1, Chunlan Wang, Yu Zhao
1Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, Hefei Anhui, 230022, P. R. China. wang-wxy@126.com
Objective:
To construct a replication-detective recombinant adinovirus including tne target gene human bone morphogenetic protein 4 (fragment hBMP-4).
Methods:
The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/ hBMP-4 was transformed into the packaging of the cell lines HEK293 to produce the replication-defective recombinant adenoviruses containing the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay.
Results:
The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by the Western-blot assay.
Conclusion:
The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
