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Cloning, sequencing, expression and inflammatory activity in skin of ovine interleukin‐8
Author(s) -
SEOW HF.,
YOSHIMURA T.,
WOOD P. R.,
COLDITZ I. G.
Publication year - 1994
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1038/icb.1994.59
Subject(s) - complementary dna , microbiology and biotechnology , amino acid , affinity chromatography , peptide sequence , biology , cdna library , fusion protein , sepharose , open reading frame , molecular cloning , biochemistry , recombinant dna , gene , enzyme
Summary Ovine IL‐8 (oIL‐8) cDNA was obtained by probing a spleen cell cDNA library with human IL‐8 (hIL‐8) cDNA. The oIL‐8 cDNA was 1434 base pairs long with a single open reading frame encoding a 101 amino acid precursor protein of relative molecular mass 11 268. The inferred amino acid sequence has 78, 82, 84 and 67% similarity with human, rabbit, porcine and guinea‐pig IL‐8, respectively. By analogy with the most prevalent form of hIL‐8, a 72 amino acid form of oIL‐8 was expressed as a fusion protein containing glutathione‐ S ‐transferase and purified by affinity chromatography on a glutathione‐Sepharose column yielding 8 mg IL‐8/L broth culture. The fusion protein lacked chemotactic activity for ovine neutrophils, whereas the 72 amino acid form of oIL‐8 was equipotent with rhIL‐8. At 6 and 24 h after intradermal injection of 10 −1 mol oIL‐8, there was intense accumulation of neutrophils, and very mild accumulation of eosinophils, CD5, CD4 and T19 (a σδ5 TCR subset) cells but not CD8 cells. The availability of roIL‐8 and its cDNA probes will permit the role of this important member of the IL‐8 family of chemotactic cytokines to be determined in inflammatory diseases of sheep.