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  • Title: Binding of divalent H-2Kd/IgG2aFc fusion protein to murine macrophage via Fc-FcR interaction.
    Author: Xiao W, Chen X, Lin Zhou, Lu S, Liang Z, Wu X.
    Journal: Cell Mol Immunol; 2007 Apr; 4(2):147-51. PubMed ID: 17571461.
    Abstract:
    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 (Mphi) through its Fc portion binding to Fc receptor (FcR) of Mphi. 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 supernatant with a Staphylococcal Protein A (SPA) column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western 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 Mphi 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 Mphi, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine Mphi, which can be applied to activate antigen-specific cytotoxic T lymphocyte (CTL).
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