Cloning and characterisation of an ovine interleukin-10-encoding cDNA.
Expression of the interleukin 10-encoding (IL-10) mRNA by ovine (ov-) cells, in response to mitogenic stimulation, was assessed by Northern blot and polymerase chain reaction (PCR) analyses using a human (hu) IL-10 cDNA probe and oligodeoxyribonucleotide primers based on homologous regions of the human and murine IL-10 cDNA sequences. A 315-bp cDNA generated by the PCR analysis was cloned and used to screen a lipopolysaccharide-stimulated alveolar ov-macrophage cDNA library. The full-length ov-cDNA sequence isolated translates to a protein of 177 amino acids (aa) with a predicted 18-aa leader sequence and molecular mass of 20,165 Da. Expression in a mammalian system demonstrated that the ov-cDNA encoded a protein with the expected IL-10 biological activity. Both recombinant huIL-10 and supernatants from COS cells transfected with an expression vector containing the ovIL-10 cDNA inhibited production of IL-1 and tumour necrosis factor-alpha by ov-alveolar macrophages. Genomic DNA analysis indicated ovIL-10 exists as a single gene within the ov-genome.
Martin
H M
HM
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Australia.
Nash
A D
AD
Andrews
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis; D018076:DNA, Complementary / Q000235:genetics; D016678:Genome; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep / Q000235:genetics*; D013045:Species Specificity; D014018:Tissue Distribution
D016207:Cytokines; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016753:Interleukin-10