Molecular cloning and identification of macrophage migration inhibitory factor (MIF) in teleost fish.

Abstract

Macrophage migration inhibitory factor (MIF) is one of the first cytokines to be identified, which have been emerged to be an important mediator of the innate and adaptive immune system. Although MIF was well characterized in several mammal species, there was still little report in fish. In present study, we cloned the MIF gene from Tetraodon nigroviridis, and identified other six MIF genes from other teleost fishes, Fundulus heteroclitu, Oncorhynchus mykiss, Ictalurus punctatus, Danio rerio, Salmo salar and Haplochromis chilotes. The results showed that the fish MIF genes with the same organization as the mammalians consist of three exons and two introns. Tetraodon MIF gene located within a 1091bp genomic fragment of chromosome 1, transcribed into a 500bp mRNA including 14bp 5' untranslated region (UTR), 348bp ORF and 138bp 3'-UTR. Tetraodon MIF with 115aa has a calculated molecular mass of 12.5kDa and a theoretical pI of 6.81. The deduced amino-acid sequences of the teleost fish MIFs showed 64.1-73.5% sequence identity to mammalian MIFs, 61.5-70.1% to avian MIFs, 55.6-62.4% to amphibian MIFs, 74.4-97.4% among the teleost fishes. Phylogenetic analysis separates the teleost fish MIFs into an exclusive group. Genomic Southern blotting analyses suggest that Tetraodon has one copy of the MIF gene. RT-PCR and real-time PCR analyses reveal that Tetraodon MIF (TnMIF) mRNA was constitutively expressed in 10 selected tissues and induced by lipopolysaccharide (LPS) strikingly in head kidney and spleen. The bioactivity of recombinant TnMIF was tested by macrophage migration inhibition (MMI) assay. The result of MMI assay showed that the recombinant TnMIF inhibited the macrophage cells migration at rate of 35% (P<0.04). These results indicated that MIFs in fish may be involved in immune responses.