Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro.
OBJECTIVE
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 [(3)H]-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.


CONCLUSIONS
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.
Mao
Yi-Xiang
YX
Institute of Biotechnology, Soochow University, Suzhou 215007, China.
Chen
Yong-Jing
YJ
Ge
Yan
Y
Ma
Hong-Bing
HB
Yu
Jian-Feng
JF
Wu
Hong-Ya
HY
Hu
Yu-Min
YM
Wang
Qin
Q
Shi
Qin
Q
Zhang
Xue-Guang
XG
D018122:B7-1 Antigen / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology*; D014161:Transduction, Genetic; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1
D018122:B7-1 Antigen; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1; C475826:VTCN1 protein, human; D007371:Interferon-gamma; D005982:Glutathione Transferase