Acta Pharmacologica Sinica 2006 June; 27 (6): 741-746; doi: 10.1111/j.1745-7254.2006.00338.x

 
Original Article
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Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro1
 

Yi-xiang MAO2,3, Yong-Jing CHEN2, Yan GE2, Hong-bing MA2, Jian-feng YU2, Hong-ya WU2, Yu-min HU2, Qin WANG2, Qin SHI2, Xue-guang ZHANG2,4

2Institute of Biotechnology, Soochow University, Suzhou 215007, China; 3Department of Oncology, First Affiliated Hospital to Soochow University, Suzhou 215007, China

 

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-γ 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

 
1 Project supported by a grant from the Major State Basic Research Development Program of China (973 Program; No 2001CB51003).

4 Correspondence to Prof Xue-guang ZHANG.
Phn/Fax 86-512-6510-4908.
E-mail smbxuegz@publicl.sz.js.cn
Received 2005-10-23     Accepted 2006-01-06

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