In vitro and in vivo bioactivity of single-chain interleukin-12.
Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.
Foss
D L
DL
University of Minnesota, Department of Veterinary PathoBiology, St. Paul, MN 55108, USA.
Moody
M D
MD
Murphy
K P
KP
Pazmany
C
C
Zilliox
M J
MJ
Murtaugh
M P
MP
D000276:Adjuvants, Immunologic; D000284:Administration, Oral; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006801:Humans; D007114:Immunization; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000276:immunology; D007583:Jejunum / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000378:metabolism; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D015398:Signal Transduction; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013552:Swine
D000276:Adjuvants, Immunologic; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12; D007371:Interferon-gamma