Fish Interleukin-4 Gene Cloning And Expression Analysis

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-a. Much work on 1L-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, and obtained recombinant IL-4 (rIL-4) expressed in Pichia pastoris. 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 2μg ConA, 2μg PHA, and 2μg 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. Recombinant expression plasmid pPIC9k-IL4 was successfully constructed, and transformed into P. pastoris strain GS115. Transformants were screened with auxotrophic medium MD and G418. Induced with methanol, the predicted expression of rIL-4 was detected. This is the first report of cloning, characterization and expression of IL-4 in fish.