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目的构建编码增强型绿色荧光蛋白(green fluorescent protein,EGFP)和人CD40L胞外段(hecdCD40L)组成的融合基因真核表达载体,并探讨表达后的融合蛋白对人树突状细胞(dendritic cells,DCs)表型和功能的影响。方法以RT-PCR方法从健康人外周血单个核细胞中克隆hecdCD40L基因片段。小鼠血管内皮细胞生长因子受体2信号肽序列(SigmKDR)和EGFP与hecdCD40L基因片段融合或不融合,插入真核表达载体pcDNA3.1(-),构建pcDNA3.1SigmKDR-EGFP(sEGFP)和pcDNA3.1sEGFP-hecdCD40L。用脂质体将表达载体转染至体外培养的COS-7细胞后,G418抗性筛选,流式细胞仪分选并检测EGFP和/或CD40L的表达。Western blot检测融合蛋白在COS-7细胞及其上清中的表达,用镍柱和超滤离心管获取纯化浓缩的sEGFP-hecdCD40L和sEGFP融合蛋白与人DCs共孵育,流式细胞仪和ELISA分析DCs对2种融合蛋白的摄取率、DCs表型变化以及细胞因子IL-12的分泌。结果 pcDNA3.1sEGFP或pcDNA3.1sEGFP-hecdCD40L转染COS-7细胞后,经G418抗性筛选和流式细胞仪分选筛选获得高效表达融合蛋白的单克隆细胞株,EGFP阳性细胞可达95%以上,Westernblot在细胞内和上清中均能检测到融合蛋白。sEGFP或sEGFP-hecdCD40L与DCs共孵育2h,流式细胞仪检测示EGFP阳性率前者为28.6%,后者为56.9%,24h后分析DCs的表型,sEGFP-hecdCD40L融合蛋白增强DCs表面CD80、CD83和HLA-DR分子的表达以及刺激IL-12的分泌,而sEGFP对DCs的表型和IL-12的分泌无明显影响。结论用SigmKDR、EGFP和hecdCD40L构建的融合基因能在真核细胞中表达并能分泌至细胞外,这种分泌型融合蛋白能靶向DCs,诱导DCs的成熟和上调其表面共刺激分子和MHC-II分子的表达,并刺激IL-12分泌。
Objective To construct a fusion gene eukaryotic expression vector encoding enhanced green fluorescent protein (EGFP) and human CD40L extracellular domain (hecdCD40L) and to investigate the effect of the expressed fusion protein on dendritic cells (dendritic cells, DCs) phenotype and function. Methods The hecdCD40L gene fragment was cloned from human peripheral blood mononuclear cells by RT-PCR. The mouse vascular endothelial growth factor receptor 2 signal peptide sequence (SigmKDR) and EGFP hecdCD40L gene fragment fusion or non-fusion, insert the eukaryotic expression vector pcDNA3.1 (-), construct pcDNA3.1SigmKDR-EGFP (sEGFP) and pcDNA3 .1sEGFP-hecdCD40L. After transfection of the expression vector to COS-7 cells cultured in vitro with liposomes, G418 resistance screening, flow cytometry sorting and detection of EGFP and / or CD40L expression were performed. Western blot was used to detect the expression of fusion protein in COS-7 cells and its supernatant. Purified and purified sEGFP-hecdCD40L and sEGFP fusion proteins were obtained by nickel column and ultrafiltration centrifuge tube and incubated with human DCs. Flow cytometry and ELISA DCs uptake of two fusion proteins, phenotypic changes of DCs and secretion of cytokine IL-12. Results After transfection of pcDNA3.1sEGFP or pcDNA3.1sEGFP-hecdCD40L into COS-7 cells, the McAbs with high expression of fusion protein were obtained by G418 resistance screening and flow cytometry screening. The EGFP-positive cells reached more than 95% , Western blot can detect the fusion protein both in the cell and in the supernatant. sEGFP or sEGFP-hecdCD40L were co-cultured with DCs for 2h. The positive rate of EGFP was 28.6% and the latter was 56.9% by flow cytometry. The phenotypes of DCs were analyzed after 24h. sEGFP-hecdCD40L fusion protein enhanced the expression of CD80, CD83 And HLA-DR molecules and stimulate the secretion of IL-12, while sEGFP had no significant effect on the phenotypes of DCs and the secretion of IL-12. Conclusion The fusion gene constructed by SigmKDR, EGFP and hecdCD40L can be expressed in eukaryotic cells and secreted to the outside of cells. The secreted fusion protein can target DCs, induce the maturation of DCs and up-regulate the surface costimulatory molecules and MHC- II molecules and stimulate IL-12 secretion.