[Construction and identification of replication-defective recombinant adinovirus expressing human bone morphogenetic protein 4].
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.


CONCLUSIONS
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.
Wang
Xingyu
X
Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, Hefei Anhui, 230022, P. R. China. wang-wxy@126.com
Wang
Chunlan
C
Zhao
Yu
Y
D000256:Adenoviridae / Q000235:genetics*; D055415:Bone Morphogenetic Protein 4 / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D003673:Defective Viruses / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering; D014162:Transfection
D055415:Bone Morphogenetic Protein 4