Jiang Yao, He Libo, Ju Changsong, Pei Yongyan, Ji Myonghuan, Li Yongming, Liao Lanjie, Jang Songhun, Zhu Zuoyan, Wang Yaping
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; University of Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Fish Shellfish Immunol. 2015 May;44(1):88-99. doi: 10.1016/j.fsi.2015.01.024. Epub 2015 Feb 7.
Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio rerio. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-κB, IRF7, IL-1β, and TNF-α also up-regulated significantly, whereas the IκB gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish.
Toll样受体5（TLR5）是Toll样受体（TLRs）家族的成员之一，负责脊椎动物中细菌鞭毛蛋白的识别，在先天免疫中发挥重要作用。在本研究中，克隆并分析了草鱼（Ctenopharyngodon idellus）的两个TLR5基因，命名为CiTLR5a和CiTLR5b。CiTLR5a和CiTLR5b均为典型的TLR蛋白，包括富含亮氨酸重复序列（LRR）基序、跨膜区和TIR结构域。CiTLR5a的全长cDNA为3054 bp，开放阅读框（ORF）为2646 bp，5'非翻译区（UTR）为78 bp，3'UTR为330 bp。CiTLR5b的全长cDNA为3326 bp，ORF为2627 bp，5'UTR为95 bp，3'UTR为594 bp。系统发育分析表明，CiTLR5a和CiTLR5b与鲮鱼、鲤鱼和斑马鱼的TLR5亲缘关系较近。亚细胞定位表明，CiTLR5a和CiTLR5b具有相似的定位模式，可能定位于转染细胞的质膜上。实时定量PCR显示，CiTLR5a和CiTLR5b在所有检测组织中均组成性表达，但最高表达组织有所不同。在暴露于鞭毛蛋白和草鱼呼肠孤病毒（GCRV）后，CiTLR5a和CiTLR5b显著上调。此外，TLR5信号通路的下游基因如髓样分化因子88（MyD88）、核因子κB（NF-κB）、干扰素调节因子7（IRF7）、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）也显著上调，而IκB基因下调，表明CiTLR5a和CiTLR5b参与了对鞭毛蛋白刺激和GCRV感染的应答。本研究结果将为进一步了解TLR5在硬骨鱼类中的功能提供新的见解。
