Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems

• Published in: Equine veterinary journal Vol. 33; no. 7; pp. 693 - 698
• Main Authors: McMONAGLE, E. L. J., TAYLOR, S., ZUILEKOM, H. van, SANDERS, L., SCHOLTES, N., KEANIE, L. J., HOPKINS, C. A., LOGAN, N. A., BAIN, D., ARGYLE, D. J., ONIONS, D. E., SCHIJNS, V. E., NICOLSON, L.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-11-2001
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Baculoviridae; Cell Line; cytokine; DNA, Complementary - genetics; Gene Expression Regulation; Genetic Vectors; horse; Horse Diseases - immunology; Horse Diseases - therapy; Horses - genetics; Horses - immunology; IL-12; Immunity, Cellular; interferon-gamma; Interferon-gamma - biosynthesis; interleukin; Interleukin-12 - biosynthesis; Interleukin-12 - chemistry; Interleukin-12 - genetics; Interleukin-12 - immunology; Interleukin-12 Subunit p35; Interleukin-12 Subunit p40; Molecular Sequence Data; Protein Subunits; Spodoptera; Th1 cytokine
• ISSN: 0425-1644; 2042-3306
• DOI: 10.2746/042516401776249426

Summary
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‐γ production in cells derived from equine lymph nodes. Preincubation of IFN‐γ inducing preparations with anti‐p40 monoclonal antibody resulted in a significant decrease in IFN‐γ induction capacity. Medium recovered from p35 and p40‐expressing baculovirus infected cultures enhanced target cell IFN‐γ 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.