Molecular Cloning And Expression Analysis Of The IL-1β Gene In Mystus Macropterus

As an important member of the interleukin1family, interleukin-1β (IL-1β) is one of the early pivotal response pro-inflammatory cytokines that induce inflammation by mediating a series of reactions and enhance anti-infection of organism. Studies showed that many stimulating factors can lead to change expression of the IL-1β gene, and suggested that the IL-1β might play an important role in physiological processes against diseases in fish. Mystus macropterus, an economic fish species in the Yangtze River, suffered from bacterial diseases. In order to investigate the IL-1β’ s effect of the M.macropterus against diseases, the IL-1β gene expression has been studied in this paper.In this study, the IL-1β gene in M. macropterus has been cloned with RT-PCR and RACE-PCR methods. It contains1194nucleotides, including3′UTR of224nucleotides,5’UTR of82nucleotides, and an open reading frame with888nucleotides encoding296amino acid peptides with MW of33.84kD and PI of5.00. Conserved signature sequences of IL-1gene family were founded in the M.macropterus IL-1β amino acid sequence which had been deduced. Analysis with the Signal P software revealed that there was no signal peptide in the sequence, which was in common with the other known IL-1β molecules. Just as other nonmammalian IL-1β genes sequenced to date, the M.macropterus IL-1β was lack of an aspartic acid in cut region of mammalian IL-1β which was required for cleavage by ICE (interleukin-1converting enzyme). Compared with eight vertebrates, IL-1β in M.macropterus shared the highest identity (79.0%) with that in Ictalurus punctatus, and the lowest identity (22.0%) with that in Gallus gallus. Phylogenetic tree analysis based on some IL-1β amino acids indicated that IL-1β amino acid in M.macropterus was clustered closely with that from I. punctatus.Semi-quantitative RT-PCR was used to analyze tissues distribution of IL-1β mRNA expression in unstimulated M.macropterus. Analysis with tissues distribution of IL-1β mRNA indicated that IL-1β transcriptional expression levels were detectible in all tissues (muscle, brain, spleen, head kidney, kidney, stomach, skin, liver and intestine), but highest expression in head kidney and spleen, lowest in brain.After injection with Aeromonas hydrophila, an increased level of IL-1β expression was obversed in head kidney and spleen cells compared to that of unstimulated cells. Expression of head kidney and spleen began to increase until4h after stimulation. The IL-1β expression of head kidney and spleen showed a peak level on day1and recovered to the normal level on day15after stimulation.M.macropterus is more sensitive to the stimulus of lipopolysaccharide (LPS) than the stimulus of A hydrophila. After2hour injection with LPS, the IL-1β expression of head kidney and spleen began to increase significantly after injection for2hours, and showed a peak level in head kidney and spleen at4hours after injection with LPS, and recovered the normal level at7days.The results indicated that M.macropterus IL-1β was sensitive to stimulus of A. hydrophila or LPS, and suggested IL-1β might play a critical role in the process against the dieseas caused by Gram negative bacteria.