Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: The first evidence of Th2-type cytokine in fish

Interleukin-4 (IL-4) is one of the key cytokines in Th2 mediated immune responses, which has been shown to regulate the responses of many immune cytokines, such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and TNF-α. Much work on IL-4 has been done in human and several mammal species while little in fish. In this study, we have cloned and characterized the full-length cDNA of IL-4 in Tetraodon. The Tetraodon IL-4 cDNA is 834 bp in length and contains a short 5′UTR of 39 bp, a 3′UTR of 375 bp and an open reading frame of 420 bp translating into a protein of 139 aa with a predicted molecular mass of 16.131 kDa. The Tetraodon IL-4-encoding gene with the same organization as the mammalians and birds consists of four exons and three introns. The encoded protein shows 11–16% identities to other homologues. RT-PCR was optimized to estimate the expression level of IL-4 in Tetraodon. The results showed that IL-4 is constitutively expressed in all selected tissues, including head kidney, spleen, liver, brain, gill, muscle and heart, although low levels were observed in head kidney, spleen, and liver. The ubiquitous expression of IL-4 is consistent with a postulated role in immune cytokines regulation. Stimulating the fish with a mixed stimulant that contained 2 μg ConA, 2 μg PHA, and 2 μg PMA, significantly up-regulated the expression of IL-4 in most tissues examined, which potentially indicated that IL-4 was involved in the immune inflammatory responses triggered by mitogens. This is the first report of cloning and characterization of IL-4 cDNA and gene in fish.