[Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells].
OBJECTIVE
To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro.


METHODS
Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR.


RESULTS
The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC.


CONCLUSIONS
The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.
Luo
Ling-hui
LH
Department of Otorhinolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Gong
Shu-sheng
SS
Song
Peng
P
Yan
Kai-sheng
KS
Liu
Ying-peng
YP
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D005801:Genes, Homeobox; D019254:Genes, bcl-2; D005822:Genetic Vectors; D006801:Humans; D051381:Rats; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic; D013136:Spiral Ganglion / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes