Human recombinant B7-H3 expressed in E. coli enhances T lymphocyte proliferation and IL-10 secretion in vitro.
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.
Zhang
Guang-Bo
GB
Institute of Biotechnology, Soochow University, Key Laboratory of Clinical Immunology of Jiangsu Province, Suzhou 215007, China.
Chen
Yong-Jing
YJ
Shi
Qin
Q
Ma
Hong-Bing
HB
Ge
Yan
Y
Wang
Qin
Q
Jiang
Zhi
Z
Xu
Ying
Y
Zhang
Xue-Guang
XG
D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD; D060887:B7 Antigens; D018122:B7-1 Antigen / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D060887:B7 Antigens; D018122:B7-1 Antigen; C423237:CD276 protein, human; D004396:Coloring Agents; D016207:Cytokines; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D016753:Interleukin-10; D007544:Isopropyl Thiogalactoside; D007371:Interferon-gamma; D005982:Glutathione Transferase; C022616:thiazolyl blue