[Construction of recombinant adenovirus vector carrying the mouse caveolin-1 in gene].
OBJECTIVE
To construct recombinant adenovirus carrying the mouse dentin caveolin-1 gene using the recombinant adenoviral vector system AdEasy.


METHODS
The cDNA fragment of caveolin-1 was derived from pTRE2-caveolin-1 by restriction enzyme digestion and subcloned into shuttle plasmid pAdtrack-CMV. The resulting plasmid pAdtrack-CMV-caveolin-1, after linearized by digesting with restriction endonuclease Pme I, was transformed into E. coli 1 BJ5183 which had been transformed by adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-caveolin-1 was screened by kanamycin LB plate and then identified by restriction enzyme digestion. The linearized adenovirus plasmid pAd-caveolin-1 was packaged in 293 cells, then the recombinant adenovirus Ad-caveolin-1 was harvested. The expression of green fluorescence protein was observed under fluorescent microscope. With further amplification and purification, the titer of recombinant adenovirus was determined.


RESULTS
The recombinant adenovirus was identified by restriction enzyme digestion analysis and gene sequencing. Cytopathic effect and the expression of GFP were observed in the infected 293 cells. The sequence of caveolin-1 gene insert was the same as that published in the GenBank. The titer of the recombinant adenovirus was 2 x 10(9) pfu/mL.


CONCLUSIONS
The mouse caveolin-1 recombinant adenovirus was constructed successfully by using AdEasy adenovirus system. Cell transfection and expression of exogenous gene can be detected directly by observing the expression of GFP. These results provide the basis for the further study on the role of caveolin-1 gene in other scopes.
Yang
Lin
L
State key Laboratory of Oral Diseases, West China Stomatological Hospital, Sichuan University, Chengdu 610041, China.
He
Yong-Hong
YH
Shen
Yong-Dai
YD
Liu
Lei
L
Tang
Wei
W
Zheng
Xiao-Hui
XH
Tian
Wei-Dong
WD
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D051242:Caveolin 1 / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D051242:Caveolin 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins