C型凝集素受体Langerin的细胞表达模型构建及功能研究  

Establishment and functional analysis of a cell model expressing the C-type lectin receptor Langerin

作　　者：张晓宁[1] 张宇[1] 罗阳[1] 李巍[2] 姚煦[1] 

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]第四军医大学西京皮肤医院

出　　处：《中华皮肤科杂志》2015年第6期408-412,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81371735）,2014年度亚美医学基金会（MMAAP）皮肤病项目基金

摘　　要：目的 构建朗格汉斯细胞特异性C型凝集素受体Langerin体外细胞表达模型.方法 PCR方法获得Langerin分子的cDNA,克隆入真核绿色荧光蛋白（EGFP）表达载体pEGFP-C1（EGFP位于Langerin的N末端）,转染人肾胚细胞癌HEK 293T细胞后,激光共聚焦显微镜检测EGFP-Langerin融合蛋白的细胞表达情况,流式细胞仪检测Langerin受体的表达,并进一步检测转染表达的Langerin受体对尘螨抗原（nDerp 2）的识别和内吞情况.结果 构建的荧光融合蛋白重组表达质粒转染HEK 293T细胞后,经PCR及Western印迹检测证明Langerin转染及表达成功.重组表达质粒转染HEK293T细胞后,经流式细胞仪检测转染Langerin融合质粒的HEK293T细胞的Langerin受体表达率较转染前增多约43％,经激光共聚焦显微镜检测显示绿色荧光标记的Langerin成功表达,并可与红色荧光素标记的尘螨抗原（nDerp 2）结合,将nDer p 2内吞入细胞内.结论 构建的EGFP-Langerin融合蛋白在HEK293T细胞内表达后具有细胞表面受体分子的特征性分布,具有结合、内吞过敏原功能.Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro.Methods The cDNA of Langerin was obtained by PCR and cloned into a eukauotic green fluorescent protein （EGFP） expression vector pEGFP-C 1 with EGFP located in the N terminal region of the Langerin gene.Then,the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T.Subsequently,laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein,and flow cytometry to measure the expression of Langerin.Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen （nDer p 2） by Langerin.Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells.As flow cytometry revealed,the expression level of Langerin in transfected HEK293T cells was increased by 43% compared with untransfected cells.Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed,and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2.Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors,and could bind to and endocytose allergens.

关 键 词：树突细胞 受体 有丝分裂原 重组融合蛋白质类 尘螨科 HEK293细胞 受体 模式识别 

分 类 号：R55[医药卫生—血液循环系统疾病]