Construction and identification of a eukaryotic expression plasmid encoding murine CD40Ig gene

Objective:To construct a eukaryotic expression vector encoding murine CD40Ig gene and make preparation for the gene therapy of induction of donor-specific immunologic tolerance.Methods:The cDNA of the extracellular domain of murine CD40 gene and IgG2a Fc gene were amplified by RT-PCR from the total RNA isolated from the mouse spleen,and the green fluorescent protein(GFP) gene was amplified by PCR with the plasmid pEGFP-N1 as the template.Then the genes were inserted into the eukaryotic expression vector pDC516,the resultant recombinant plasmid was confirmed by seqencing.Then the recombinant plasmid was transfected into human embryonic kidney cell 293(HEK293) using Lipofectamine 2000,the expressed fusion protein was observed by fluorescence microscopy.Results:The eukaryotic expression plasmid pDC516-CD40-IgG-GFP was successfully constructed,and it could be correctly expressed in HEK293 cells.Conclusion:The successfully construction of the eukaryotic expression plasmid pDC516-CD40-IgG-GFP has laid the foundation for the study of induction of donor-specific immunologic tolerance.