Structural and functional homology between duck and chicken interferon-gamma

Abstract

The Duck interferon gamma (DuIFN-γ) cDNA was cloned from a phytohaemaglutinin-stimulated duck spleen cDNA library screened using a chicken IFN-γ (ChIFN-γ) cDNA probe. The DuIFN-γ cDNA is 1392 nt long and shows 99% and 80% sequence identity with another cloned DuIFN-γ cDNA, and with ChIFN-γ cDNA, respectively. The cDNA contains a 495 bp ORF that encodes a putative 164 amino acid (AA) protein that shares 67% identity with ChIFN-γ, but only 30–35% identity with mammalian IFN-γ. The predicted three-dimensional (3D) structures of DuIFN-γ and ChIFN-γ are similar when analysed by comparative protein modelling. Culture supernatant collected from COS cells transfected with DuIFN-γ cDNA was able to activate nitrite secretion from a chicken macrophage cell line (HD11) in a dose-dependent fashion. This activity could not be neutralised by an anti-ChIFN-γ monoclonal antibody (Mab 85) that was able to neutralise the activity of ChIFN-γ. Recombinant DuIFN-γ (rDuIFN-γ) protein was expressed in E. coli as an N-terminally His-tagged protein and was purified on a nickel affinity column. The eluted protein, which was detected as a ∼18 kDa band with a purity of >90%, was also detected by Western blot using the anti-ChIFN-γ monoclonal antibody (Mab 9.1). The rDuIFN-γ was shown to activate nitrite secretion by HD11 cells in a dose-dependent fashion with a specific activity that was ∼16-fold lower than a rChIFN-γ control. Two rabbit antisera raised against rDuIFN-γ were able to neutralise COS cell-expressed DuIFN-γ activity; one of these also neutralised ChIFN-γ activity. These findings indicate that DuIFN-γ shares structural and functional identity with ChIFN-γ, which is consistent with our previous results which demonstrated cross reactivity with other lymphokines from the two species.