EGFP-CD40L融合基因的构建、表达及功能鉴定

Construction,expression and characteristics of EGFP-CD40L fusion gene

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 an eukaryotic expression vector encoding a fusion gene consisting of enhanced green fluorescent protein （EGFP） and human extracellular domain of CD40L （hecdCD40L）,and to investigate the effects of the expression product of the fusion gene on phenotypes and function of human dendritic cells （DCs）. Methods hecdCD40L was cloned from human peripheral blood mononuclear cells （PBMC） by RT-PCR. The signal peptide of murine vascular endothelial cell growth factor receptor-2 （SigmKDR） and EGFP were fused with or without hecdCD40L,and then the fused fragments was inserted into the eukaryotic expression vector pcDNA3.1（-） to construct pcDNA3.1 SigmKDR-EGFP （sEGFP） and pcDNA3.1 sEGFP-hecdCD40L. After transfection of COS-7 cells with the vectors by lipofectamine 2000TM,the monoclonal COS-7 cells stably expressing sEGFP or sEGFP-hecdCD40L fusion proteins were obtained via G418-resistant screening and sorting of flow cytometer （FACS）. The expression efficiencies of the fusion genes were analyzed by FACS and the fusion proteins in the cells and supernatants were detected by Western blotting,respectively. Uptake rates,phenotypic changes,and secretion of interleukin-12 （IL-12） in DCs were analyzed by FCAS and ELISA after co-incubation of DCs with the sEGFP or sEGFP-hecdCD40L proteins depurated and concentrated by Ni-NTA Agarose and ultrafiltration. Results After the transfection of the pcDNA3.1 sEGFP or pcDNA3.1 sEGFP-hecdCD40L into COS-7 cells,the monoclonal COS-7 cells expressing the fusion proteins were obtained by G418-resistant screening and flow cytometer sorting,in which the cells expressed EGFP reached up to 95% by the analysis of FACS. Western blotting showed the expression of the fusion proteins in the cells and its supernatants. In addition,uptake rates to sEGFP or sEGFP-hecdCD40L by DCs were 28.6% and 56.9%,respectively,in 2 h after co-incubation of sEGFP or sEGFP-hecdCD40L with DCs. The increased expression of CD80,CD83,and HLA-DR and secretion of IL-12 on DCs were detected by FCAS and ELISA in 24 h after co-incubation with sEGFP-hecdCD40L protein. However,the sEGFP protein had no such an effect on DCs. Conclusion The fusion protein consisting of SigmKDR,EGFP,hecdCD40L is expressed in the eukaryotic cells and secreted out of the cells. The secreting protein can be targeted to DCs,and then induce maturation of DCs,up-regulate costimulatory and MHC-Ⅱ molecules on DCs,and stimulate the IL-12 secretion of DCs.