Characterization of caprine interleukin-4.
Caprine interleukin-4 (IL-4) cDNA was cloned from RNA of mitogen stimulated goat peripheral blood mononuclear cells utilizing reverse transcriptase-polymerase chain reaction. The sequence of caprine IL-4 cDNA corresponds to a 535 nucleotide mRNA with 5'- and 3'-untranslated regions and a 405 nucleotide open reading frame, the first 66 nucleotides of which encode a putative signal peptide. Mature IL-4 is a 12.8kDa protein containing six cysteine residues and two potential N-linked glycosylation sites and is highly homologous with other ruminant IL-4. The predicted molecular mass of mature unglycosylated IL-4 was confirmed by western blot of recombinant caprine IL-4 expressed in bacteria with a monoclonal antibody against a carboxyterminal peptide derived from the predicted amino acid sequence of bovine IL-4. Eukaryotic expression plasmids containing caprine IL-4 cDNA were used to characterize recombinant IL-4. Transcription of IL-4 mRNA was confirmed by transfection of COS-7 and goat synovial membrane cells, and recombinant IL-4 produced by stably transfected L929 cells inhibited inducible nitric oxide synthase in macrophages. Genetic immunization of mice with a caprine IL-4 cDNA expression plasmid induced antibodies against recombinant caprine IL-4 produced in bacteria.
Snekvik
K R
KR
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA. ksnek@vetmed.wsu.edu
Beyer
J C
JC
Bertoni
G
G
Von Beust
B R
BR
Baszler
T V
TV
Palmer
G H
GH
McElwain
T F
TF
Cheevers
W P
WP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006041:Goats / Q000235:genetics* / Q000378:metabolism; D015847:Interleukin-4 / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015847:Interleukin-4