Cloning, expression and functional analysis of a novel-chemokine gene of Japanese flounder, Paralichthys olivaceus, containing two additional cysteines and an extra fourth exon

A CC chemokine gene (JFCCL3) was cloned and sequenced from Japanese flounder, Paralichthys olivaceus.The JFCCL3 cDNA contains an open reading frame of 288 nucleotides encoding 95 amino acid residues. The predicted amino acid sequence of JFCCL3 showed the conserved cysteine of the β chemokine plus two additional cysteines. The genomic sequence consists of two isoforms: JFCCL3.1 and JFCCL3.2 with sizes of 1.8 and 1.2 kb, respectively. Both isoforms contain three introns and four exons. RT–PCR showed that JFCCL3 is constitutively expressed in most tissues including lymphoid organs. Using quantitative real-time RT–PCR, the highest expression of JFCCL3 transcripts was observed in PBLs at 3 h post-stimulation with Con A/PMA and at 1 h post-stimulation with LPS. A phylogenetic analysis showed that JFCCL3 is more closely related to fractalkines than to other mammalian β chemokines. A chemotaxis assay showed that recombinant JFCCL3 protein has bioactivity for Japanese flounder leukocyte attraction at concentrations from 0.01 to 10 μg/ml.