isopropyl-thiogalactoside and thiazolyl-blue

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.

Topics: Antibodies, Monoclonal; Antigens, CD; B7 Antigens; B7-1 Antigen; Blotting, Western; Cell Line; Cell Proliferation; Cloning, Molecular; Coloring Agents; Cytokines; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Genetic Vectors; Glutathione Transferase; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-10; Isopropyl Thiogalactoside; Protein Structure, Tertiary; Receptors, Immunologic; Recombinant Fusion Proteins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tetrazolium Salts; Thiazoles