中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (7): 457-460.

• 论著 • 上一篇    下一篇

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

张宇1,姚煦2,顾汉艳1,王宝玺3,刘军4   

  1. 1. 中国医学科学院皮肤病医院
    2. 南京 中国医学科学院北京协和医学院皮肤病研究所
    3. 中国医学科学院北京协和医学院整形外科医院
    4. 南京大学医学院附属鼓楼医院皮肤科
  • 收稿日期:2013-09-02 修回日期:2014-03-24 发布日期:2014-07-01
  • 通讯作者: 姚煦 E-mail:dryao_xu@126.com
  • 基金资助:
    国家自然科学基金;江苏省自然科学基金

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

  • Received:2013-09-02 Revised:2014-03-24 Published:2014-07-01
  • Supported by:
    ;Natural Science Foundation of Jiangsu Province of China

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-SIGN N末端,转染人胚肾细胞癌 HEK 293T 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内吞入细胞内。 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。

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

Abstract: Zhang Yu*, Yao Xu, Gu Hanyan, Wang Baoxi, Liu Jun. *Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Yao Xu, Email: dryao_xu@126.com; Liu Jun, Email: drliu_jun@126.com 【Abstract】 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 cells, 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.

Key words: Receptors, mitogen, Recombinant fusion proteins, Antigens, dermatophagoides, Cell line, tumor

引用本文

张宇 姚煦 顾汉艳 王宝玺 刘军. 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[J]. 中华皮肤科杂志, 2014,47(7):457-460. doi: