大口黑鲈MyD88基因的克隆及其对NF-κB的激活作用  

Cloning of Largemouth Bass(Micropterus salmoides)MyD88 Gene and Its Activating Effect on NF-κB

作　　者：高风英[1] 董浚键[1] 张赫桐 李佳鑫 朱芷琳 孙成飞[1] 叶星[1] GAO Feng-Ying;DONG Jun-Jian;ZHANG He-Tong;LI Jia-Xin;ZHU Zhi-Lin;SUN Cheng-Fei;Ye Xing(Pearl River Fisheries Research Institute/Key Laboratory of Tropical&Subtropical Fishery Resource Application&Cultivation,Ministry of Agriculture,Chinese Academy of Fishery Science,Guangzhou 510380,China;College of Fisheries,Tianjin Agricultural University,Tianjin 300384,China)

机构地区：[1]中国水产科学研究院珠江水产研究所/农业部热带亚热带水产资源利用与养殖重点实验室,广州510380 [2]天津农学院水产学院,天津300384

出　　处：《农业生物技术学报》2025年第2期366-378,共13页Journal of Agricultural Biotechnology

基　　金：中国水产科学研究院基本科研业务费(2023TD95);现代农业产业技术体系专项资金(CARS-46)。

摘　　要：接头蛋白髓细胞分化因子88(myeloid differentiation factor 88,MyD88)通常参与白介素-1受体(interleukin-1 receptor,IL-1R)和Toll样受体(Toll-like receptor,TLR)介导的核因子κB(nuclear factor kappa-B,NF-κB)激活,在先天免疫中发挥重要作用。在本研究中,克隆了大口黑鲈(Micropterus salmoides)MyD88的全长cDNA,包含一个867 bp的ORF,编码288个氨基酸残基的肽。大口黑鲈MyD88的推测蛋白具有N末端死亡结构域和C末端TIR(Toll-like/IL-1)结构域,已知这些结构域是哺乳动物MyD88的重要功能结构域。大口黑鲈MyD88蛋白与其他脊椎动物具有较高的同源性(58.5%~99.3%)。系统发育分析显示,大口黑鲈MyD88与其他鱼类MyD88聚为一支。MyD88 mRNA在健康大口黑鲈的所有被检测组织中均有表达,在肝脏中表达量最高。为了探索大口黑鲈MyD88的先天免疫作用,研究了其在肠、鳃、脾、肾中,对聚胞肌酸(polyinosinic-polycytidylic acid,PolyI:C)和诺卡氏菌(Nocardia seriolae)刺激的基因表达谱。结果显示,在所有4种检测的组织中,2种免疫刺激剂均上调了大口黑鲈MyD88转录水平,其中受诺卡氏菌的诱导最强。肾组织中,MyD88在感染后9 d出现了最大上调,感染组表达量为对照组的8.5倍(P<0.05);在鳃组织中PolyI:C诱导启动最早,刺激后8 h,在鳃组织中实验组相对表达量为对照组的5.6倍。大口黑鲈MyD88分布于HeLa细胞质中。大口黑鲈MyD88的过表达可以激活NF-κB。这些结果为阐明MyD88在大口鲈鱼先天免疫中的作用提供了基础数据。Adapter protein myeloid differentiation factor 88(MyD88)is involved in the interleukin-1 receptor(IL-1R)and Toll-like receptor(TLR)-mediated activation of nuclear factor-kappaB(NF-κB),which plays important role in innate immunity.In this study,the full-length cDNA of largemouth bass(Micropterus salmoides)MyD88 was isolated.Its ORF was 867 bp in length which encoded 288 amino acid residues.Protein secondary structure analysis showed that MyD88 protein had a N-terminal death domain and a TIR(Toll-like/IL-1)domain,which are known as important functional structural domains in mammalian MyD88.The largemouth bass MyD88 protein had high identity(58.5%~99.3%)with other vertebrates.Phylogenetic analysis showed that largemouth bass MyD88 gathered together with MyD88 of other fish species.In healthy largemouth bass,MyD88 mRNA was detected in all sampled tissues,and MyD88 mRNA had the highest expression levels in the liver.To study the role of MyD88 in innate immunity,its mRNA expression profile after stimulation with polyinosinic-polycytidylic acid(PolyI:C)and Nocardia seriolae was studied in the intestine,gill,spleen and kidney.The results showed that in all 4 detected issues,2 kinds of immune stimuli both upregulated the transcription level of largemouth bass MyD88,and the induction by N.seriolae in the kidney was the strongest.In kidney tissue,MyD88 showed the maximum upregulation at 9 d after infection,and the expression level in the infection group was 8.5 times of that in the control group(P<0.05).The induction by PolyI:C in the gill was initiated earliest.In gill tissue,after 8 h of stimulation,the relative expression level of the experimental group was 5.6 times of that in the control group.Largemouth bass MyD88 were distributed in the HeLa cytoplasm.Overexpression of MsMyD88 could activate NF-κB.These results provide basic data for elucidating the role of MyD88 in the innate immunity of largemouth bass.

关 键 词：大口黑鲈 髓细胞分化因子88(MyD88) 克隆 表达分析 亚细胞定位 核因子κB(NF-κB) 

分 类 号：S917.4[农业科学—水产科学]