Lee Dae-Sim, Hong Su Hee, Lee Hyun-Jeong, Jun Lyu Jin, Chung Joon-Ki, Kim Ki Hong, Jeong Hyun Do
Department of Aquatic Life Medicine, Pukyong National University, 599-1 Dae Yeon Dong, Nam Ku, Busan 608-737, South Korea.
Comp Biochem Physiol A Mol Integr Physiol. 2006 Mar;143(3):307-14. doi: 10.1016/j.cbpa.2005.12.014. Epub 2006 Jan 31.
The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.
利用从已知鱼类白细胞介素-1β（IL-1β）序列设计的引物进行聚合酶链反应（PCR），随后使用cDNA末端快速扩增法（RACE）对尼罗罗非鱼（Oreochromis niloticus）的IL-1β全长cDNA序列进行了测定。该cDNA包含一个92个碱基对的5'非翻译区（UTR）、一个732个碱基对的单一开放阅读框（ORF），其编码一个243个氨基酸的分子、一个341个碱基对的3'UTR，带有四个细胞因子RNA不稳定基序（ATTTA），以及在poly(A)尾上游15个核苷酸处的一个聚腺苷酸化信号（AATAAA）。基于该cDNA序列的基因组IL-1β的结构似乎是4个内含子和5个外显子。与已知的IL-1β氨基酸序列（包括人、猫鲨、鳟鱼、大菱鲆、鲤鱼、海鲷、海鲈和金鱼）相比，从尼罗罗非鱼cDNA序列推导的氨基酸序列显示出不同程度的同一性，范围从25.32%到66.80%，同源性范围从41.88%到82.19%。尽管尼罗罗非鱼IL-1β的整个cDNA序列与其他已报道的IL-1β cDNA显示出49.45%到67.05%的同一性，但每个外显子与其他已报道的IL-1β cDNA的相应外显子也显示出不同程度的同一性。尼罗罗非鱼IL-1β的外显子1和外显子2 - 5的最高核苷酸序列同一性分别在海鲷和海鲈的相应外显子中发现。在用脂多糖（LPS）进行体外刺激后，我们发现头肾细胞中IL-1β的表达水平比未刺激的细胞有所增加。然而，在刺激4小时后这种差异不再明显，此时刺激细胞和未刺激细胞中的水平相似。通过腹腔注射LPS在体内刺激的头肾细胞在1天后显示出IL-1β表达的峰值水平，在3天后表达水平下降。在刺激7天后，我们几乎无法检测到IL-1β的表达。
