Porcine interleukin-12 fusion protein and interleukin-18 in combination induce interferon-gamma production in porcine natural killer and T cells.
A pcDNA3 vector containing a gene encoding a porcine interleukin-12 (poIL-12) fusion protein was constructed, with the p40 chain and its signal peptide positioned first, followed by a linker and the p35 domain. When expressed in COS cells, secreted poIL-12 fusion protein showed high activity in terms of ability to induce interferon-gamma (IFN-gamma) production in porcine peripheral blood mononuclear cells (PBMCs) in vitro. The IFN-gamma production induced by poIL-12 fusion protein, as well as heterodimeric poIL-12 and human IL-12, was markedly dependent on the presence of human IL-18 (huIL-18). Furthermore, huIL-18 showed a dose-dependent induction of IFN-gamma production in PBMC in the presence of a constant concentration of huIL-12. A marked synergism between poIL-12 and IL-18 was consequently observed in poPBMC. The actual IFN-gamma producing cells were identified as probable NK cells (about 30%) and T lymphocytes (about 70%), using flow cytometry. Furthermore, a histidine-tagged poIL-12 fusion protein was expressed in Drosophila melanogaster Schneider 2 cells, using a modified pMT/V5-His vector lacking the V5 epitope. Such poIL-12 fusion protein was easily purified using Ni-NTA agarose and retained high biological activity.
Domeika
K
K
Department of Veterinary Microbiology, Section of Immunology, Swedish University of Agricultural Sciences, Biomedical Centre, P.O. Box 588, SE-75123, Uppsala, Sweden. kristina.domeika@vmm.slu.se
Berg
M
M
Eloranta
M-L
ML
Alm
G V
GV
D000818:Animals; D001681:Biological Assay; D019556:COS Cells; D002460:Cell Line; D004331:Drosophila melanogaster; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000008:administration & dosage* / Q000235:genetics; D020382:Interleukin-18 / Q000008:administration & dosage*; D007694:Killer Cells, Natural / Q000187:drug effects* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics; D013552:Swine / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology*
D020382:Interleukin-18; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12; D007371:Interferon-gamma