Abstract
To explore the biofunctions of human B7-H3 on activated T lymphocyte, the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase (GST) fusion protein. A 49 kD fusion protein (named as GST/hB7-H3 hereafter) was induced by IPTG and purified by standard methods reported in prokaryotic system. In the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was observed by incubating purified T cells with soluble GST/hB7-H3 fusion protein by MTT assay. The concentrations of IFN-gamma and IL-10 in the supernatants of T cells were determined by ELISA. The results showed that the GST/hB7-H3 protein produced in bacteria had modest biological activities to proliferate the T lymphocyte and enhance IFN-gamma as well as IL-10 secretion.
MeSH terms
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Antibodies, Monoclonal / chemistry
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Antigens, CD
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B7 Antigens
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B7-1 Antigen / chemistry*
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B7-1 Antigen / metabolism
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Blotting, Western
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Cell Line
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Cell Proliferation
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Cloning, Molecular
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Coloring Agents / pharmacology
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Cytokines / metabolism
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli / metabolism
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Genetic Vectors
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Glutathione Transferase / metabolism
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Humans
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In Vitro Techniques
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Interferon-gamma / metabolism
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Interleukin-10 / biosynthesis*
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Interleukin-10 / metabolism
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Isopropyl Thiogalactoside / pharmacology
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Protein Structure, Tertiary
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Receptors, Immunologic
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / chemistry*
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Recombinant Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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T-Lymphocytes / cytology*
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T-Lymphocytes / metabolism
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Tetrazolium Salts / pharmacology
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Thiazoles / pharmacology
Substances
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Antibodies, Monoclonal
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Antigens, CD
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B7 Antigens
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B7-1 Antigen
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CD276 protein, human
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Coloring Agents
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Cytokines
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Receptors, Immunologic
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Recombinant Fusion Proteins
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Recombinant Proteins
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Tetrazolium Salts
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Thiazoles
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Interleukin-10
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Isopropyl Thiogalactoside
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Interferon-gamma
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Glutathione Transferase
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thiazolyl blue