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Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro

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Abstract:

Abstract

Aim: To explore the biofunctions of human B7-H4 generated from prokaryotic system. Methods: The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [3H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. Results: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. Conclusion: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.

Keywords: B7-H4; glutathione s-transferase fusion protein; inhibition

Document Type: Research Article

DOI: http://dx.doi.org/10.1111/j.1745-7254.2006.00338.x

Affiliations: Institute of Biotechnology, Soochow University, Suzhou 215007, China

Publication date: June 1, 2006

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