Structural and functional homology between duck and chicken interferon-gamma.
The Duck interferon gamma (DuIFN-gamma) cDNA was cloned from a phytohaemaglutinin-stimulated duck spleen cDNA library screened using a chicken IFN-gamma (ChIFN-gamma) cDNA probe. The DuIFN-gamma cDNA is 1392 nt long and shows 99% and 80% sequence identity with another cloned DuIFN-gamma cDNA, and with ChIFN-gamma cDNA, respectively. The cDNA contains a 495 bp ORF that encodes a putative 164 amino acid (AA) protein that shares 67% identity with ChIFN-gamma, but only 30-35% identity with mammalian IFN-gamma. The predicted three-dimensional (3D) structures of DuIFN-gamma and ChIFN-gamma are similar when analysed by comparative protein modelling. Culture supernatant collected from COS cells transfected with DuIFN-gamma 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-gamma monoclonal antibody (Mab 85) that was able to neutralise the activity of ChIFN-gamma. Recombinant DuIFN-gamma (rDuIFN-gamma) 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 approximately 18 kDa band with a purity of >90%, was also detected by Western blot using the anti-ChIFN-gamma monoclonal antibody (Mab 9.1). The rDuIFN-gamma was shown to activate nitrite secretion by HD11 cells in a dose-dependent fashion with a specific activity that was approximately 16-fold lower than a rChIFN-gamma control. Two rabbit antisera raised against rDuIFN-gamma were able to neutralise COS cell-expressed DuIFN-gamma activity; one of these also neutralised ChIFN-gamma activity. These findings indicate that DuIFN-gamma shares structural and functional identity with ChIFN-gamma, which is consistent with our previous results which demonstrated cross reactivity with other lymphokines from the two species.
Huang
A
A
Department of Microbiology and Immunology, University of Adelaide, Australia.
Scougall
C A
CA
Lowenthal
J W
JW
Jilbert
A R
AR
Kotlarski
I
I
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D019556:COS Cells; D002478:Cells, Cultured; D002645:Chickens / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004372:Ducks / Q000378:metabolism*; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021281:Quantitative Structure-Activity Relationship; D011817:Rabbits; D017422:Sequence Analysis, DNA / Q000662:veterinary
D018076:DNA, Complementary; D007371:Interferon-gamma