Alpha-7 nicotinic acetylcholine receptor (alpha 7nAChR) played an important role during the process of inflammation, and was a potential therapeutic target for many diseases, including Alzheimer's disease, endotoxaemia, hypertension etc. However, it was difficult to express alpha 7nAChR on commonly used host cells because of the lack of the chaperone protein RIC-3, which was indispensable to promote folding, assembly, and surface expression of alpha 7nAChR. This work was designed to develop a cell line, which not only expressed alpha 7nAChR highly and stably, but possessed biological activity. Full-length alpha 7nAChR gene was extracted from rat peritoneal macrophages, recombinant plasmid pIRES2-EGFP-alpha 7nAChR was constructed, and then transfected to the mouse fibroblast cell line NIH3T3, a cell line expressing RIC-3. Compared with the NIH3T3-pIRES2-EGFP, expression of alpha 7nAChR was significantly higher in NIH3T3-pIRES2-EGFP-alpha 7nAChR on both mRNA and protein levels. To evaluate the anti-inflammation activity of the over-expressed alpha 7nAChR, TNF-alpha, IL-6, and IL-1 beta release induced by lipopolysaccharide was determined. No changes were found between NIH3T3-pIRES2-EGFP and NIH313-pIRES2-EGFP-alpha 7nAChR culture supernatant under LIPS challenge, however under nicotine pre-stimulation, pro-inflammatory cytokine release was significantly attenuated in alpha 7nAChR transfected cell line. Moreover, in this established cell line, PNU-282987, a selective alpha 7nAChR agonist, exerted a stronger ability to reduce TNF-a release than nicotine. We concluded that a functional NIH3T3-pIRES2-EGFP-alpha 7nAChR cell line was developed, which might be useful for agonist screen and biological research. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.