Molecular characterization, recombinant expression and bioactivity analysis of the interleukin-1β from the yellowfin sea bream, Acanthopagrus latus (Houttuyn)

Abstract Interleukin-1β (IL-1β) is an important inflammatory mediator and has also the potential as an immunoadjuvant. Here we describe the isolation and characterization of yellowfin sea bream IL-1β (sbIL-1β) cDNA and gene. The sbIL-1β cDNA contains a 121-bp 5′ untranslated region (UTR), a single open reading frame (ORF) of 762 bp that translated into a 253 amino acid protein, a 342-bp 3′ UTR with six cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail. The organization of the genomic IL-1β appears to be five exons and four introns, the intron and exon boundaries all follow the GT-AG consensus. The analysis of the expression pattern showed that sbIL-1β was expressed weakly in the kidney, spleen, gill and intestine, but not in the liver, heart and muscle. After injection with 150 μg LPS, the expression analysis in vivo showed that sbIL-1β was induced in the kidney and spleen and expression level was maximal after 4 h. The predicted 253 amino acid sequence shares 23.5–88.5% identity and 43.9–93.3% similarity to known IL-1β. Thus, IL-1β is very conserved in fish of the same family. No interleukin-converting enzyme (ICE) cut site is found in sbIL-1β, but the alignment of the amino acid sequence with other species showed a possible cut site between Tyr 87 and Thr 88 that would give rise to a 166-amino-acid mature peptide. The putative mature peptide was expressed in E. coli , and the recombinant sbIL-1β could induce the transcription of sbIL-1β in a dose-dependent manner and the expression levels were almost equal in the samples treated with 50 ng/ml recombinant sbIL-1β and 5 μg/ml LPS, which showed recombinant sbIL-1β was biologically active and had the potential as an immunoadjuvant.