小鼠CD40Ig基因真核表达质粒的构建及鉴定_医学论文.doc免费

小鼠CD40Ig基因真核表达质粒的构建及鉴定_医学论文 小鼠CD40Ig基因真核表达质粒的构建及鉴定_医学论文 【摘要】 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。 【关键词】 CD40Ig 质粒 真核表达载体 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. 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. Key words: CD40Ig plasmid eukaryotic expression vector 器官移植是当今治疗终末期脏器衰竭的有效方法，但免疫排斥是器官移植后长期生存的一大难题。T细胞是介导排异反应的主要细胞，CD40/CD40L通路在T细胞的激活过程中发挥着重要作用，因此阻断CD40/CD40L间的相互作用，可以诱导宿主对移植物的低排斥反应性。由此我们克隆了小鼠CD40分子胞外区和IgG2a分子Fc段的cDNA，并以pEGFP-N1质粒为模板进行PCR，获得GFP基因，经酶切连接插入真核表达载体pDC516，制备出重组质粒pDC516-CD40-IgG-GFP，转染293细胞后见GFP表达，证实质粒pDC516-CD40-IgG-GFP构建成功，从而为下一步构建含CD40Ig基因的腺病毒载体作好了准备，为研究阻断CD40/CD40L通路诱导宿主对移植物的低排斥反应性奠定了基础。 1 材料和方法 1.1 实验动物 Balb-c小鼠