Binding of Divalent H-2Kd/IgG2aFc Fusion Protein to Murine Macrophage via Fc-FcR Interaction

• 作 者: Wei Xiao Xueling Chen Lin Zhou
• 机 构: Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan
• 出 处: 中国免疫学杂志
• 发表日期: 2007年2期
• 关键词: H-2Kd/IgG2aFc divalent MHC fusion protein Fc receptor
• 基金项目: 国家自然科学基金

Peptide-MHC class Ⅰ complex (pMHC) is a specific ligand for TCR recognition, and important for CD8+T cell activation. Here we described a genetically engineered divalent class Ⅰ major histocompatibility complex (MHC) molecule, H-2Kd/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2Kd, a murine MHC class Ⅰ molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2Kd molecule to the surface of murine macrophage (Mφ) through its Fc portion binding to Fc receptor (FcR) of Mφ. cDNAs coding for the extracellular domains of H-2Kd and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1(+). The H-2Kd/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supatant with a Staphylococcal Protein A (SPA) column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and West blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2Kd heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2Kd indicated the fusion protein consists of both Fc portion and H-2Kd. Peritoneal Mφ of C57BL/6 (H-2Kb) can be stained with H-2Kd specific monoclonal antibody (mAb) after incubated with the H-2Kd/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2Kd molecule to the surface of murine Mφ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine Mφ, which can be applied to activate antigen-specific cytotoxic T lymphocyte (CTL).