中文摘要：

目的构建携带鼠可溶性CD40分子（sCD40）及增强型绿色荧光蛋白（EGFP）报告基因的融合蛋白真核表达质粒pEGFP．N1／sCD40并检测其在树突状细胞（DC2．4）中的表达水平。方法采用基因工程技术构建CD40胞外区与EGFP重组载体质粒pEGFP—N1／sCD40，经酶切及序列分析鉴定，脂质体介导转染DC2．4细胞株，荧光显微镜，荧光分光光度计及SDS。PAGE检测sCD40-EGFP融合蛋白的表达，流式细胞仪检测其活性。结果融合基因在瞬时转染的树突状细胞中获得表达，并分泌至上清，且sCD40-EGFP融合蛋白中分子标签未影响可溶性sCD40膜外活性区的生物学活性。结论可溶性CD40分子与EGFP融合基因载体构建成功，在树突状细胞中表达的融合蛋白具有生物活性，为进一步研究基因修饰树突状细胞诱导特异性移植免疫耐受奠定了实验基础。

英文摘要：

[ Abstract] Objective To construct a recombinant plasmid carrying enhanced green fluorescent protein（EGFP） and mouse soluble CD40 gene （sCD 40） and detect its expression levels in dendritic Cells （DC 2.4 ）. Methods The standard cloning technology was employed to construct the EGFP/ VEGF fusion gene which was identified with double enzyme digestion and DNA sequencing, then this re- combinant plasmid was transfected into mouse dendritic Cells （DC2.4） with lipofectamine. The expres- sion levels of the fusion gene were detected by fluorescence microscope, fluorescence spectrophotometer, SDS-PAGE, and flow cytomerey （FCM）, respectively. Results The fusion gene was observed in transiently transfected dendritic cells. The fusion protein was secreted into su- pernatant. These data suggested that the EG-FP tag did not interfere with the natural assembly and the biological activity of soluble CD40. Con- clusions The recombinant plasmid carrying enhanced green fluorescent protein and mouse soluble CD40 gene is successfully constructed and expressed positively in rat DCs with activity.