[Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].
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.
Wang
Wei-rong
WR
Department of Pharmacology, Medical School of Xi'an Jiaotong University, Xi'an 710061, China. wangweirong1222@163.com
Lin
Rong
R
Yang
Yu-cong
YC
Gan
Wei-jie
WJ
Liu
Jun-tian
JT
Lü
She-min
SM
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
D019013:CD40 Antigens; D008081:Liposomes; D011994:Recombinant Proteins; D023201:CD40 Ligand