Identification, characterization and the interaction of Tollip and IRAK-1 in grass carp (Ctenopharyngodon idellus)
Identification, characterization and the interaction of Tollip and IRAK-1 in grass carp (Ctenopharyngodon idellus)
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草鱼 (Ctenopharyngodon idellus) 中 Tollip 和 IRAK-1 的鉴定、表征及其相互作用
DOI：
10.1016/j.fsi.2012.05.025
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发表时间：
2012-09-01
影响因子：
4.7
通讯作者：
Wang, Yaping
中科院分区：
文献类型：
--
作者：
Huang, Rong;Lv, Jianjian;Wang, Yaping
Tollip和IRAK-1是哺乳动物TLR/IL-1R信号转导通路的关键组分，作为TLR/IL-1R信号转导通路的介导剂发挥重要作用。虽然已经在鱼类中发现了几种TLR，但在鱼类中感染诱导的免疫中的分子关联、蛋白质蛋白质相互作用或TLR信号通路的作用很少受到关注。本研究从草鱼头肾cDNA文库中克隆了Tollip和IRAK-1基因序列。分别通过3 '和5' RACE和基因组步移获得全长转录本和启动子区序列。利用构建的报告基因-启动子和实时荧光定量RT-PCR技术分析草鱼Tollip和IRAK-1基因在组织中的转录模式。重组蛋白用于抗体生产。在系统发育树上，草鱼与已报道的Tollip和IRAK-1基因聚为一类，与斑马鱼同源性最高。草鱼Tollip和IRAK-1的启动子区被证明是有活性的。病毒感染后，这两个基因座的转录水平在大多数免疫相关组织中以时间依赖性方式上调。免疫荧光分析表明Tollip和IRAK-1在CIK细胞的胞质中均匀分布并共定位。然而，在病毒感染后，Tollip和IRAK-1都趋向于细胞膜。我们的研究结果表明，存在的Tollip和IRAK-1蛋白质的硬骨鱼类，并建议Tollip-IRAK-1复合物被招募到受体复合物刺激后，病毒。这些结果提供了新的见解TLR信号通路的作用，硬骨鱼类，特别是行动的硬骨鱼Tollip和IRAK-1和这些分子的相互作用，作为这一途径的一部分。(C)2012爱思唯尔有限公司保留所有权利。
Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study. Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway. (C) 2012 Elsevier Ltd. All rights reserved.