Identification and molecular cloning of functional chicken IL-12.
By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.
Degen
Winfried G J
WG
Department of Vaccine Technology and Immunology, Intervet International BV, Boxmeer, The Netherlands. winfried.degen@Intervet.com
van Daal
Nancy
N
van Zuilekom
Hanneke I
HI
Burnside
Joan
J
Schijns
Virgil E J C
VE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D002645:Chickens / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000166:cytology / Q000276:immunology
D018076:DNA, Complementary; C508858:IL12A protein, human; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018664:Interleukin-12; D007371:Interferon-gamma