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Title: Cloning, Expression and Biological Analysis of Recombinant Chicken IFN-gamma Expressed in Escherichia coli

Author
item Lillehoj, Hyun
item LI, GUANGXING - Northeast Agricultural University
item ZENG, YAN - Northeast Agricultural University
item REN, XIAOFENG - Northeast Agricultural University

Submitted to: Hybridoma
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2010
Publication Date: 6/10/2010
Citation: Lillehoj, H.S., Li, G., Zeng, Y., Ren, X. 2010. Cloning, Expression and Biological Analysis of Recombinant Chicken IFN-gamma Expressed in Escherichia coli. Hybridoma. 29(1):1-6.

Interpretive Summary: Cytokines are soluble hormone-like factors which are secreted by activated lymphocytes. One example is interferon-gamma (IFN-') which has been used to measure host cell-mediated immunity in various animal species. In this study, ARS scientist collaborated with scientists at the College of Veterinary Medicine, Northeast Agricultural University in China to develop mouse monoclonal antibodies that detect chicken IFN-'. They isolated chicken gene encoding IFN-' from a local White Leghorn chicken, and recombinant chicken IFN-' was expressed in E.coli. These antibodies specifically recognized chicken IFN-' and will be a useful reagent as a diagnostic tool to assess host immunity in chickens. Availability of these reagents will facilitate progress in basic and applied immunological research in poultry.

Technical Abstract: The interferon-gamma (CHIFN-') derived from the spleen cells of White Leghorns chicken, a local Chinese breeding species was amplified by RT-PCR. The gene encoding CHIFN-' with the deletion of the N-terminal signal peptide was cloned into prokaryotic expression vector pET30a, resulting in a recombinant plasmid pET-30a-CHIFN-'. After the pET-30a-CHIFN-' was transformed into host cells BL21(DE3)pLysS, the expression of CHIFN-' was induced by Isopropyl ß-D-thiogalactoside (IPTG) at 37'. Rabbit antisera were raised using the soluble CHIFN-' as immunogen. Immunoreactivities of the CHIFN-' and its antisera were investigated by immunoblotting and ELISA. Moreover, the antiviral effect of the CHIFN-' was analyzed. Our data indicate that the CHIFN-' is biologically active. Keywords: Interferon-gamma; Expression; Biological activity; Polyclonal antisera