香鱼白细胞介素-1受体相关激酶3(IRAK-3)的序列特征及对鳗弧菌感染的免疫相关性分析  

Sequence Characteristics of Interleukin-1 Receptor-Related Kinase 3(IRAK-3) in Ayu(Plecoglossus altivelis) and Its Immune Correlation Analysis Against Vibrio anguillarum Infection

作　　者：李尚阳 周燕[1,2] 柴方超 周前进 陈炯[1,2] LI Shang-Yang;ZHOU Yan;CHAI Fang-Chao;ZHOU Qian-Jin;CHEN Jiong(State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products,Ningbo University,Ningbo 315211,China;School of Marine Science,Ningbo University,Ningbo 315832,China)

机构地区：[1]宁波大学农产品质量安全危害因子与风险防控国家重点实验室,宁波315211 [2]宁波大学海洋学院,宁波315832

出　　处：《农业生物技术学报》2020年第10期1711-1721,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972821);浙江省自然科学基金重点项目(LZ18C190001);浙江省公益技术研究计划/农村农业(LGN18C180002);宁波市自然基金(2018A610342);宁波市科技创新团队(2015C110018);宁波市江北区农业和社会发展科技计划项目(2017B02);浙江省近岸水域生物资源开发与保护重点实验室2018年开放基金(J2018004);2018年浙江省大学生科技创新活动计划暨新苗人才计划(2018R405078)。

摘　　要：白细胞介素-1受体相关激酶3 (interleukin-1 receptor-associated kinase-3, IRAK-3)是IRAK蛋白家族的成员,在多种感染性及非感染性疾病中,可通过抑制促炎因子的产生而调节免疫自稳和免疫耐受。IRAK-3在鱼类感染与免疫应答进程中的研究鲜有报道。本研究从香鱼(Plecoglossus altivelis)单核/巨噬细胞转录组中获得了香鱼的IRAK-3基因(PaIRAK-3),分析发现其开放阅读框为1 722 bp,预测编码573个氨基酸,理论分子量为63.2 kD,拥有IRAK家族特有的N末端死亡结构域(N-terminal death domain)、富含脯氨酸/丝氨酸/苏氨酸结构域(proline/serine/threonine-rich domain, ProST domain)、苏氨酸丝氨酸激酶结构域(kinase and/or pseudokinase domain)和C末端结构域(C-terminal domain)。系统进化树的结果显示,IRAK-3在鱼类中相对保守,PaIRAK-3与虹鳟(Oncorhynchus mykiss)和银大麻哈鱼(Oncorhynchus kisutch)的IRAK-3同源性最高,分别为63.1%和63.0%。q RT-PCR分析结果表明,PaIRAK-3主要在肾脏、鳃、脾脏、肝脏及肠组织中表达;鳗弧菌(Vibrio anguillarum)感染后,香鱼肾脏、脾脏及肝脏中的PaIRAK-3的mRNA水平明显上调,均在感染8 h达到最大值;鳃组织中PaIRAK-3仅在8 h呈现明显上调。同时,本研究在大肠杆菌(Escherichia coli) BL21 Rosetta中表达了重组蛋白PaIRAK-3 (recombinant PaIRAK-3, rPaIRAK-3),利用纯化的rPaIRAK-3免疫小鼠(Mus musculus),制备了多克隆抗体。利用该抗体进行的Western blot分析结果表明,鳗弧菌感染后,肝脏和脾脏组织中PaIRAK-3的表达量明显上调,在12 h达到最大值。综上,本研究从mRNA水平和蛋白质水平上解析了PaIRAK-3与香鱼的鳗弧菌感染紧密相关,为深入探讨PaIRAK-3在抗菌免疫中的具体分子机制提供了参考依据。Interleukin-1 receptor-associated kinase-3(IRAK-3) is a member of the IRAK family. It can regulate immune homeostasis and tolerance in a number of infectious and non-infectious diseases through its inhibition of pro-inflammatory cytokine production. Recently, few studies are focusing on the function of IRAK-3 in fish. Herein, an IRAK-3 transcript(PaIRAK-3) was obtained from the monocyte/macrophage transcriptome of ayu(Plecoglossus altivelis). It possessed an open reading frame of 1 722 bp, predictedly encoding 573 amino acids, and had a theoretical molecular weight of 63.2 kD. PaIRAK-3 exhibited a Nterminal death domain, a proline/serine/threonine-rich domain, a kinase and/or pseudokinase domain and a Cterminal domain of IRAK family. The phylogenetic tree showed that IRAK-3 was relatively conservative in fish, and PaIRAK-3 had the highest homology with rainbow trout(Oncorhynchus mykiss)(63.1%) and silver salmon(Oncorhynchus kisutch)(63.0%). Quantitative PCR analysis showed that PaIRAK-3 was mainly expressed in the kidney, gill, spleen, liver and intestine. After Vibrio anguillarum infection, PaIRAK-3 was significantly up-regulated at the mRNA level in the kidney, spleen and liver of ayu, reaching the maximum value at 8 h post infection. And the m RNA level of PaIRAK-3 increased only at 8 h post infection in the gill tissue. Recombinant PaIRAK-3(r PaIRAK-3) was prokaryotically expressed using BL21 Rosetta strain of Escherichia coli, and the purified r PaIRAK-3 was used to immunize mice(Mus musculus) to prepare the polyclonal antibodies. Western blot analysis using the polyclonal antibodies against rPaIRAK-3 showed that PaIRAK-3 was significantly up-regulated against V. anguillarum infection at the protein level in the liver and spleen tissue and reached the maximum value at 12 h post infection. In conclusion, PaIRAK-3 is closely related to V. anguillarum infection, which provides a premise for further research on the antibacterial immunity of PaIRAK-3.

关 键 词：白细胞介素-1受体相关激酶3(IRAK-3) 香鱼 鳗弧菌 免疫 

分 类 号：S917.4[农业科学—水产科学]