DC-SIGN荧光融合蛋白的构建、表达和生物学功能初探

目的 构建绿色荧光蛋白（EGFP）标记的DC-SIGN分子（DC-SIGN-EGFP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DC-SIGN分子和EGFP分子的cDNA，分别克隆入真核表达载体pEGFP-N1和真核表达载体pCI，使EGFP荧光蛋白分别位于DC-SIGN蛋白的C末端和N末端。在转染COS-7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COS-7细胞后绿色荧光蛋白位于细胞膜，仅EGFP位于DC-SIGNN-末端的融合蛋白可被抗DC-SIGN抗体结合。EGFP位于DC-SIGN N-末端的融合蛋白可有效摄取DC-SIGN受体的高亲和力配体le（x）寡糖。结论 所构建的DC-SIGN-EGFP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DC-SIGN抗体检测及摄取特异性配体，为进一步深入研究DC-SIGN分子功能提供了良好的模型。

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

Objective To investigate the expression and function of EGFP-DC-SIGN fused in different terminals 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.