title: [Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].
abstract: OBJECTIVE
  To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells.
  
  
  METHODS
  The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively.
  
  
  RESULTS
  Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry.
  
  
  CONCLUSIONS
  The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.
authors.affiliation: Department of Pharmacology, Medical School of Xi'an Jiaotong University, Xi'an 710061, China. wangweirong1222@163.com
authors.forename: Wei-rong
authors.initials: WR
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Lin
authors.forename: Yu-cong
authors.initials: YC
authors.lastname: Yang
authors.forename: Wei-jie
authors.initials: WJ
authors.lastname: Gan
authors.forename: Jun-tian
authors.initials: JT
authors.lastname: Liu
authors.forename: She-min
authors.initials: SM
authors.lastname: Lü
mesh_terms: D019013:CD40 Antigens / Q000096:biosynthesis / Q000235:genetics*; D023201:CD40 Ligand / Q000096:biosynthesis / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
chemical_list: D019013:CD40 Antigens; D008081:Liposomes; D011994:Recombinant Proteins; D023201:CD40 Ligand