斜带石斑鱼TLR3基因的克隆及其在刺激隐核虫感染时的表达分析

MOLECULAR CLONING AND CHARACTERIZATION OF A TLR3 FROM EPINEPHELUS COIOIDES INFECTED WITH CRYPTOCARYON IRRITANS

TLR3(Toll like receptor 3)是Toll样受体家族的重要成员,通过识别病原相关分子模式,诱导宿主天然免疫应答。研究从斜带石斑鱼(Epinephelus coioides)中克隆得到TLR3 cDNA序列,全长为2937 bp,包括107bp的5′非编码区、100 bp的3′非编码区和编码909个氨基酸的2730 bp的开放阅读框。TLR3全长氨基酸序列包含1个信号肽、18个富含亮氨酸的重复序列(Leucine-rich Repeat LRR)、1个跨膜结构域和1个胞内TIR结构域(IL-R1 homologous region)。同源比对显示,斜带石斑鱼TLR3与其他已报道硬骨鱼类的TLR3具有较高的同源性(52%—67%)。组织表达分析显示,TLR3在健康斜带石斑鱼的组织中具有较广的表达分布,其中在前脑、体肾和脾脏中表达量较高。刺激隐核虫(Cryptocaryon irritans)感染斜带石斑鱼后:在皮肤中TLR3的表达量呈现先降低后升高的趋势,从感染后第7天开始上调,并在第10天达到高峰;而在脾脏中,TLR3的表达量在感染6h时就显著上调并达到峰值。结果表明斜带石斑鱼TLR3在抗刺激隐核虫的免疫应答过程中可能发挥重要作用。

Toll-like receptor （TLR）, one family of I type trans-membrane protein, is a kind of well-studied pattern recognition receptors （PRRs） recognizing pathogen-association molecular patterns （PAMPs）. Mammalian TLR3 is best-known for its recognition of pathogen dsRNA and triggering various innate immune responses against virus infec- tion. In contrast to mammalian TLR3, piscine TLR3 can detect not only virus but also bacterial PAMPs. However, whether piscine TLR3 also involved in host anti-parasite immunity is still poorly understood. In the present study, we first cloned an Epinephelus coioides TLR3 （EcTLR3） cDNA sequence by homology cloning and RACE technique. The full length of EcTLR3 was 2937 bp including a 5＇-terminal untranslated region （UTR） of 107 bp, a 3＇-terminal untrans- lated region （UTR） of 100 bp and an open reading frame （ORF） of 2730 bp encoding a polypeptide of 909 amino acid residues. The putative isoelectric point （pI） and molecular weight （Mw） of EcTLR3 was 8.27 and 102.68 kD, respec- tively. The TLR family motifs, such as leucine-rich repeat （LRR） domains and Toll/interleukin-1 receptor （TIR） domain were also conserved in EcTLR3, with 18 LRR domains and one TIR domain which were connected by one trans-membrane domain. The multiple sequence alignment demonstrated that the full length EcTLR3 amino acid se- quence shared high percentage of identities with other teleost （52%--67%）, and a somewhat higher sequence identity with the TIR domains （from 55% to 72%）, showing that the TIR domain was more highly conserved than that of other domains during TLR3 evolution. To better understand the evolutionary relationship of EcTLR3, a phylogenetic tree was constructed using reported TLR3 protein sequence. The tree indicated that EcTLR3 was branched into the same cluster as that of other teleost TLR3, and showed a highly correlated evolutionary relationship with pufferfish TLR3. To deter- mine the tissues expression pattern of EcTLR3, we examined EcTLR3 expression in healthy grouper by semi-quantitative RT-PCR. The results showed that the EcTLR3 was broadly expressed in all tested tissues, while the expression levels differed noticeably among the tissues, with strong expression in the spleen, trunk kidney and prosencephalon, which imply that EcTLR3 may play an important role in host innate immune response. To identify whether EcTLR3 involved in host anti- Cryptocaryon irritans immune responses, we detected the expression change of EcTLR3 in the skin and spleen at different time points （6h, ld, 2d, 3d, 5d, 7d, 10d and 14d） post parasite infection by real-time quantitative PCR The result showed that in the skin, the expression level of EcTLR3 was significantly down-regulated at the early time points （from day 1 to 5）, while was up-regulated from day 7 and reached the peak at day 10 post the primary infection. However, in the spleen, the expression level of EcTLR3 was immediately up-regulated and reached the peak at 6h post the parasite infection. The significant up-regulation of TLR3 expression was also observed at day 3 and 5 in the spleen post the primary infection. This result suggested that EcTLR3 may also play an important role in the host defense against C. irritans infection.