Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.
Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.
McMonagle
E L
EL
University of Glasgow Veterinary School, UK.
Taylor
S
S
van Zuilekom
H
H
Sanders
L
L
Scholtes
N
N
Keanie
L J
LJ
Hopkins
C A
CA
Logan
N A
NA
Bain
D
D
Argyle
D J
DJ
Onions
D E
DE
Schijns
V E
VE
Nicolson
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006734:Horse Diseases / Q000276:immunology / Q000628:therapy; D006736:Horses / Q000235:genetics / Q000276:immunology*; D007111:Immunity, Cellular; D007371:Interferon-gamma / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D008969:Molecular Sequence Data; D021122:Protein Subunits; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D021122:Protein Subunits; D018664:Interleukin-12; D007371:Interferon-gamma