Ovine interleukin-12: analysis of biologic function and species comparison.
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.
De Rose
R
R
Cooperative Research Centre for Vaccine Technology Unit, CSIRO Animal Health, Parkville, Victoria, Australia. Robert.DeRose@dah.csiro.au
Scheerlinck
J P
JP
Casey
G
G
Wood
P R
PR
Tennent
J M
JM
Chaplin
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D006041:Goats; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D018664:Interleukin-12