Mice CD40Ig eukaryotic expression plasmid and identification of.doc

 PAGE \* MERGEFORMAT 15 Mice CD40Ig eukaryotic expression plasmid and identification of [Abstract] Objective: To construct containing mouse CD40Ig eukaryotic expression vector, in order to induce donor-specific immune tolerance of the graft gene therapy as a preparation. Methods: Mouse spleen total RNA as a template for RT-PCR Cloning of murine CD40 extracellular domain cDNA, and IgG2a Fc segment cDNA; the other with pEGFP-N1 plasmid as a template to obtain green fluorescent protein (GFP) gene. Will be derived from gene fragments inserted into eukaryotic expression vector plasmid pDC516 be pDC516-CD40-IgG-GFP, DNA sequencing is correct, using liposome transfection 2000 its human embryonic kidney epithelial cells (HEK293 cells), observed under fluorescence microscope fusion protein expression. Results: The mouse CD40Ig successfully constructed eukaryotic expression plasmid pDC516-CD40-IgG-GFP, and expressed in HEK293 cells to achieve. Conclusion: The mouse CD40Ig eukaryotic expression plasmid pDC516-CD40-IgG-GFP was successfully constructed, in order to induce donor-specific immune tolerance of graft laid the foundation for the study. [Keywords:] CD40Ig plasmid eukaryotic expression vector Abstract: 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. R