Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells

Abstract

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. Conclusion The rcombinant 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.

CLC Number:

WANG Wei-rong¹, LIN Rong¹, YANG Yu-cong², GAN Wei-jie¹, LIU Jun-tian¹, LÜ She-min³. Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells[J]. Journal of Southern Medical University, 2005, 25(12): 1474-1477.

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