Molecular characterization, recombinant expression and bioactivity analysis of the interleukin-1 beta from the yellowfin sea bream, Acanthopagrus latus (Houttuyn).
Interleukin-1 beta (IL-1 beta) 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-1 beta (sbIL-1 beta) cDNA and gene. The sbIL-1 beta cDNA contains a 121-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 762bp 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-1 beta 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-1 beta was expressed weakly in the kidney, spleen, gill and intestine, but not in the liver, heart and muscle. After injection with 150 microg LPS, the expression analysis in vivo showed that sbIL-1 beta was induced in the kidney and spleen and expression level was maximal after 4h. The predicted 253 amino acid sequence shares 23.5-88.5% identity and 43.9-93.3% similarity to known IL-1 beta. Thus, IL-1 beta is very conserved in fish of the same family. No interleukin-converting enzyme (ICE) cut site is found in sbIL-1 beta, but the alignment of the amino acid sequence with other species showed a possible cut site between Tyr87 and Thr88 that would give rise to a 166-amino-acid mature peptide. The putative mature peptide was expressed in E. coli, and the recombinant sbIL-1 beta could induce the transcription of sbIL-1 beta in a dose-dependent manner and the expression levels were almost equal in the samples treated with 50 ng/ml recombinant sbIL-1 beta and 5 microg/ml LPS, which showed recombinant sbIL-1 beta was biologically active and had the potential as an immunoadjuvant.
Jiang
Shigui
S
Aquaculture and Biotechnology Division, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Science, Guangzhou 510300, China. jiangsg@21cn.com <jiangsg@21cn.com>
Zhang
Dianchang
D
Li
Jianzhu
J
Liu
Zhenxing
Z
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016678:Genome; D053583:Interleukin-1beta / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021541:Sea Bream / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D053583:Interleukin-1beta; D011994:Recombinant Proteins