Cloning and characterization of a TNF-like protein of Plecoglossus altivelis (ayu fish)

Ayu TNF cDNA contains an open reading frame of 708 bp encoding 235 amino acids. Poly adeniration (A) signal and eight AU-rich sequences were present in 858 bp 3′ UTR. Southern blot analysis indicated that ayu TNF is single-copy gene. The genomic DNA sequence of ayu TNF, consisting of four exons and three introns, was shown to be conserved well throughout evolution from fish to mammals. The amino acid sequence of ayu TNF was shown to have 32–41% of amino acid identity to other known fish TNF, and about 30% of amino acid identity to mammalian TNFs. A phylogenetic analysis based on the amino acid sequence of TNF indicated that ayu has a distinctive evolutionary path. Also, two residues of cysteine important for the formation of the three-dimensional structure were conserved in ayu TNF. For the functional analysis, ayu TNF was inserted into expression vector pCold/TF, transferred into Chaperone Competent Cells BL21 (pKJE7); this produced soluble mature ayu recombinant TNF. Ayu recombinant TNF was shown to induce respiratory burst activity from ayu kidney. The above results indicate that ayu TNF plays an important role in phylaxis, as it does in mammals.