C型凝集素受体树突细胞特异性捕获细胞间黏附分子3非整合素细胞表达模型的构建及功能研究  

A cellular model for the expression of the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin： construction and functional analysis

作　　者：张宇[1] 姚煦[1] 顾汉艳[1] 王宝玺[1] 刘军[2] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042 [2]南京大学医学院附属鼓楼医院皮肤科

出　　处：《中华皮肤科杂志》2014年第7期457-460,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171501）;江苏省自然科学基金（BK2011127）

摘　　要：目的构建C型凝集素受体树突细胞特异性捕获细胞间黏附分子3非整合素（DC—SIGN）细胞表达模型，为后续进行DC—SIGN蛋白受体功能研究提供基础。方法PCR扩增获得DC—SIGN分子的cDNA，克隆入真核表达载体P巨细胞病毒启动子-绿色荧光蛋白重组质粒（PCMV—EGFP），EGFP位于Dc—SIGNN末端，转染人胚肾细胞癌I-IEK293T细胞后，流式细胞仪检测DC—SIGN重组分子的表达，激光共聚焦显微镜检测DC—SIGN—EGFP融合蛋白的细胞定位情况，并进一步检测转染表达的DC—SIGN受体对过敏原抗原的识别和内吞过程。结果构建的荧光融合蛋白重组表达质粒，经PCR及Western印迹证明DC—SIGN表达成功；经流式细胞仪检测转染DC—SIGN融合质粒的HEK293T细胞的DC—SIGN受体表达量增多（约50％）。重组表达质粒转染293T细胞后，激光共聚焦显微镜检测显示，绿色荧光标记的DC—SIGN位于细胞膜上，可结合红色荧光素标记的过敏原抗原Derp2，并可将Derp2内吞人细胞内。结论构建的DC—SIGN—EGFP融合蛋白在293T细胞内表达后具有细胞表面受体分子的特征性分布，可被DC—SIGN特异性抗体识别并具有摄取过敏原功能，是研究DC—SIGN分子功能的理想细胞模型。Objective To establish a cellular model for the expression of the C-type lectin dendritic cell- specific intercellular adhesion molecule-3-grabbing non-integrin （DC-SIGN）, and to provide a basis for the functional analysis of DC-SIGN. Methods The cDNA of DC-SIGN was obtained via PCR, and cloned into the eukaryotic expression vector porcine cytomegalovirus-enhanced green fluorescent protein （PCMV-EGFP） with EGFP at the N terminal of DC-SIGN. Then, the recombinant PCMV-EGFP-DC-SIGN plasmid was transfected into HEK293T cells followed by the detection of DC-SIGN expression using PCR, Western blot and flow cytometry. Confocal microscopy was performed to localize the expression of DC-SIGN-EGFP and visualize the recognization and internalization of the Derp2 allergen by DC-SIGN. Results The recombinant fluorescent fusion protein-expressing plasmid was successfully constructed. Both PCR and Western blot confirmed the expression of DC-SIGN, Flow cytometry showed that the expression of DC-SIGN was increased by approximately 50% in HEK293T cells transfected with the recombinant expression plasmid compared with those untransfected. As confocal microscopy showed, the green fluorescence-labelled DC-SIGN was located on the cell membrane, which could bind to the red fluorescence-labelled antigen Derp2 and internalize it into the cells. Conclusions The recombinant DC-SIGN- EGFP fusion protein is characteristically located on the surface of 293T ceils, which can be recognized by the DC- SIGN-specific antibody and is capable of internalizing the allergen Derp2, and may serve as an ideal cell model for further studies on molecular function of DC-SIGN.

关 键 词：受体 有丝分裂原 重组融合蛋白质类 抗原 尘螨属 细胞系 肿瘤 

分 类 号：R96[医药卫生—药理学]