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 （...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-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/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-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.展开更多
OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic c...OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.展开更多
基金supported by a grant from the National Natural Science Foundation of China(NO.30271201).
文摘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-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/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-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.
基金supported by National Natural Science Foundation of China(81330081,81202596)Specialized Research Fund for the Doctoral Program of Higher Education(20123420110003)+1 种基金Program for Tackling Key Problems in Science and Technology by Anhui Province(1301042098)China Postdoctoral Science Foundation(2013M540508)
文摘OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.
