小鼠CD40Ig基因真核表达质粒的构建及鉴定

目的：构建含小鼠CD40Ig基因的真核表达载体，为诱导供体特异性移植物的免疫耐受的基因治疗作准备。方法：以小鼠脾脏总RNA为模板，以RT-PCR法克隆小鼠CD40胞外段cDNA及IgG2a Fc段cDNA；另以pEGFP-N1质粒为模板获得绿色荧光蛋白（GFP）基因。将所得基因片段插入真核表达载体pDC516中得到重组质粒pDC516-CD40-IgG-GFP，测序鉴定正确后，用脂质体2000将其转染人胚肾上皮细胞（HEK293细胞），荧光显微镜下观察融合蛋白的表达。结果：成功构建了小鼠CD40Ig基因真核表达质粒pDC516-CD40-IgG-GFP，并在HEK293细胞中实现表达。结论：小鼠CD40Ig基因真核表达质粒pDC516-CD40-IgG-GFP构建成功，为诱导供体特异性移植物免疫耐受的研究奠定了基础。

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.