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Human recombinant B7-H3 expressed in E. coli enhances T lymphocyte proliferation and IL-10 secretion in vitro
Guang-Bo Zhang1, Yong-Jing Chen, Qin Shi
1Institute of Biotechnology, Soochow University, Key Laboratory of Clinical Immunology of Jiangsu Province, Suzhou 215007, China.
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.
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.
