Binding of divalent H-2Kd/IgG2aFc fusion protein to murine macrophage via Fc-FcR interaction.
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).
Xiao
Wei
W
Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
Chen
Xueling
X
Lin Zhou
Lu
Shengjun
S
Liang
Zhihui
Z
Wu
Xiongwen
X
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006183:H-2 Antigens / Q000235:genetics / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D015202:Protein Engineering; D011961:Receptors, Fc / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D006183:H-2 Antigens; C024755:H-2K(K) antigen; D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins