异育银鲫Toll样受体3的cDNA序列分析与蛋白质高级结构预测

cDNA sequence analysis and tertiary structure prediction of Toll-like receptor 3 gene from Carassius auratus gibelio

利用RT-PCR、RACE及克隆等方法获得了异育银鲫Toll样受体3(cagTLR3)基因全长cDNA序列。该cDNA全长3170bp,5′端非编码区181bp;3′端非编码区283bp;开放阅读框(ORF)2706bp,编码901个氨基酸。经序列同源性分析显示,其编码的氨基酸序列与金鱼、斑马鱼、斑点叉尾、虹鳟和红鳍东方有较高的相似性,其相似性分别为92%、88%、79%、75%和72%,说明TLR3在长期的进化中具有较高保守性。TLR3全长序列起始21个氨基酸残基为信号肽;50至695个氨基酸残基间包含15个富含亮氨酸的重复序列(LRR);704至726个氨基酸残基为跨膜区(TM);754至897个氨基酸残基为与白介素-1受体高度同源的区域(TIR)。将异育银鲫TLR3与人和斑马鱼胞内TIR结构的氨基酸比对后发现,Phe728、Leu738、Tyr756与Arg736在TLR3的信号转导及胞内定位过程中起到关键作用。异育银鲫TLR3胞外配体结合域的三维结构呈U型,与人TLR3的相应结构十分相似,在N端具有1个二硫键,C端有2个。因LRRs上的插入序列和14个糖基化位点,异育银鲫TLR3胞外配体结合域内表面的大量β-折叠结构呈平坦的平面。

The full length cDNA of Toll-like receptor 3 were cloned from Carassius auratus gibelio gill by means of reverse transcription polymerase chain reaction （RT-PCR）and rapid amplification of cDNA ends（RACE）. It was a good data for further studying the function of TLR3 in immunity of Carassius auratus gibelio. Sequence analysis revealed a 3170 bp cDNA containing 181 bp 5＇-untranslated region,283 bp 3＇-untranslated region and 2706 bp open reading frame（ ORF） which encoded 901 amino acids with predicted molecular weight of 10 219.8 u and theroretical isoelectric point of 8.06. The deduced amino acid sequence of Carassius auratus gibelio had high similarity with that of Carassiu auratus （goldfish）, Danio rerio , Ictalurus punctatus , Oncorhynchus mykiss and Takifugu rubripes .The similarities were 92%, 88%, 79%, 75% and 72%, respectively. Molecular phylogeny tree also showed that TLR3 of Carassius auratus gibelio shared high homology with TLR3 of Carassius auratus gibelio and Danio rerio. The full-length TLR3 of Carassius auratus gibelio contained a signal peptide in the first 21 amino acid residues, 15 leucine-rich regions （LRR） from 50th to 695th amino acid residue, a transmembrane region （TM） from 704th to 726th residue, a Toll/IL-1R homologous region （TIR） from 754th to 897th residue. Alignment with amino acids sequence of TIR from Carassius auratus gibelio, Homo sapiens and Danio rerio indicated, Phe728,Leu738, Tyr756 and Arg736 of Carassius auratus gibelio played an important role both in the receptor signaling and intracellular localization. The three-dimensional structure of Carassius auratus gibelio TLR3 ectodomain （TLR3-FCD3）） had high similarity with that of human TLR3-FCD3） and showed horsehoe-shaped solenoid structure with predicted inner diameter of 42A, predicted outer diameter of 90A and predicted thickness of 35A. Carassius auratus gibelio TLR3-ECD possessed 1 disulfide bond at N-terminal and 2 at C-terminal. The extensive β3-sheet on the molecular＇ s concave surface formed a platform several modifications, including insertions in the LRRs and 14 N-linked glycosylation sites.