DC-SIGN荧光融合蛋白的构建、表达和生物学功能初探  

Preliminary study on construction, expression, and function of DC-SIGN fluorescent fusion protein

作　　者：王靖雪[1] 张小萍[1] 贾正才[1] 耿淼[1] 吴玉章[1] 

机构地区：[1]第三军医大学基础医学部全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2006年第4期370-373,共4页Immunological Journal

摘　　要：目的构建绿色荧光蛋白(EGFP)标记的DCSIGN分子(DCSIGNEGFP融合蛋白),并在哺乳动物细胞中获得功能性表达。方法PCR方法获得DCSIGN分子和EGFP分子的cDNA,分别克隆入真核表达载体pEGFPN1和真核表达载体pCI,使EGFP荧光蛋白分别位于DCSIGN蛋白的C末端和N末端。在转染COS7细胞后,在激光共聚焦显微镜和流式细胞仪检测重组分子的表达及融合蛋白在细胞定位情况。激光共聚焦显微镜检测融合蛋白的功能情况。结果获得了预期的重组表达质粒,经DNA测序证实开放阅读框正确,转染COS7细胞后绿色荧光蛋白位于细胞膜,仅EGFP位于DCSIGNN末端的融合蛋白可被抗DCSIGN抗体结合。EGFP位于DCSIGNN末端的融合蛋白可有效摄取DCSIGN受体的高亲和力配体le(x)寡糖。结论所构建的DCSIGNEGFP融合蛋白在细胞内表达后具有细胞表面受体分子的特征性分布,可被抗DCSIGN抗体检测及摄取特异性配体,为进一步深入研究DCSIGN分子功能提供了良好的模型。Objective To investigatetheexpression and function of EGFP-DC-SIGN fused in different tenninals of EGFP ORF in mammalian cells. Methods Two different vectors were constructed by fusing DC-SIGN with full length EGFP. DC-SIGN was linked at C-terminal of EGFP in expression vector pEGFP-N1 and at N-terminal of EGFP in expression vector pCI, respectively. DC-SIGN-EGFP was transfected into Cos-7 and K562 cells. The expression and function of DC-SING-EGFP were detected by confocal microscopy and flow cytometry. Results The expression plasmids of DC-SING-EGFP fused at different terminals were constructed successfully. The DC-SIGN-EGFP fusion protein was localized on cell membrane mostly. Only DC-SIGN at C-terminal of EGFP was recognized by anti-DC-SIGN monoclonal antibody, Le（x） oligosaccharides polymers, one of DC-SIGN high affinity ligands, can be uptaken by K562 cell line via EGFP-DC-SIGN fusion protein specifically. Conclusion The DC-SIGN has been fused with EGFP, and the fusion protein is shown to be localized on cell membrane and can mediate specific ligand internalization, which are similar to native DC-SIGN. This result provides an excellent molecular model for further study of DC-SIGN function.

关 键 词：C-型凝集素DC-SIGN 荧光融合蛋白 重组表达 le(x)寡糖 

分 类 号：Q78[生物学—分子生物学]