瓦氏黄颡鱼(Pelteobagrus vachellii)Toll样受体2(TLR2)基因克隆及免疫功能

摘要

为探究Toll样受体2(Toll-like receptor 2,TLR2)及下游免疫分子对瓦氏黄颡鱼(Pelteobagrus vachellii)机体的保护作用,本研究采用RT-PCR及RACE法获得瓦氏黄颡鱼TLR2全长cDNA(2611 bp),编码789个氨基酸残基,含有10个富含亮氨酸的重复序列(Leucine Rich Repeat,LRR)和Toll/IL-1R (Toll/IL-1 Receptor Domain,TIR)同源区结构域,属Ⅰ型跨膜受体.序列同源性比对发现,瓦氏黄颡鱼TLR2 cDNA与斑点叉尾鲴、鲤及虹鳟的同源性分别为78％、62％及49％.系统进化树分析表明,瓦氏黄颡鱼TLR2与斑点叉尾鲴聚为一支.qRT-PCR分析表明,TLR2 mRNA在检测的组织中均有表达,且在头肾和脾脏中表达水平显著高于其他组织(P＜0.05).嗜水气单胞菌感染能显著上调瓦氏黄颡鱼肝脏、头肾及脾脏中TLR2 mRNA表达(P＜0.05),分别在24h、48h及12h达到最大值.头肾中的TLR 2信号通路下游的髓样分化因子、半胱氨酸蛋白酶8、核转录因子kappa B、肿瘤坏死因子α、白细胞介素1β mRNA均显著上升(P＜0.05),分别在24h、12h、48h,48h和48h达到最大值.结果表明,嗜水气单胞菌感染激活了TLR2信号通路,通过上调表达,肿瘤坏死因子α,白细胞介素1β等.本研究表明,TLR2在瓦氏黄颡鱼抵御嗜水气单胞菌侵染的过程中发挥了重要的免疫作用.%Toll-like receptors (TLRs) play essential roles in innate immunity,and TLR2 is crucial to the host defense of pathogenic microbes.In this study,a 261 lbp full-length cDNA sequence of TLR2 gene from Pelteobagrus vachellii was obtained with RT-PCR and rapid amplification of cDNA ends (RACE) technique.The translated protein was composed of 789 amino acids.The putative domains included one transmembrane domain,nine leucine-rich repeats (LRRs),and one Toll/IL-1 receptor homology domain (TIR) in TLR2 of P.vachellii.Sequence comparison indicated that the TLR2 deduced from amino acid sequence of P.vachellii had an overall identity of 78％,62％ and 49％ to that of Ictalurus punctatus,Cyprinus carpio,and Oncorhynchus mykiss,respectively.Alignment of deduced amino acid sequence to other species showed that the overall structure of TLR2 was evolutionarily conserved.Phylogenetic analysis revealed that the P.vachellii TLR2 was closely related to the TLR2 in other fish.Quantitative PCR analysis showed that TLR2 was expressed in the detected tissues,with the highest level in head kidney.After Aeromonas hydrophila challenge,the TLR2 mRNA levels increased significantly (P＜0.05) to the maximum,attained at 24h in the liver,48h in the head kidney,and 12h in the spleen.Moreover,myeloid differentiation factor 88 (MyD88) (the downstream genes of TLR2),Caspase 8,and NF-κB showed significant up-regulation in 6-96h,to the maximum levels at 24h,12h,and 48h,respectively.Tumor necrosis factor t (TNFα) and interleukin-1β (IL-1β) showed significantly up-regulation in 6-192h,to the maximum levels both at 48h.The present study indicated that TLR2 could exhibit important immune responses to virus infection.