title: [Construction of recombinant adenovirus vector carrying the mouse caveolin-1 in gene].
abstract: 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.
authors.affiliation: State key Laboratory of Oral Diseases, West China Stomatological Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: He
authors.forename: Yong-Dai
authors.initials: YD
authors.lastname: Shen
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Zheng
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Tian
mesh_terms: 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
chemical_list: D051242:Caveolin 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins