七鳃鳗myd88基因的生物学特性及其下游分子的表达模式分析

髓样分化因子88 (Myeloid differentiation factor 88, MYD88)是Toll样受体(Toll-like receptor, TLR)信号通路的关键接头分子, 在先天性免疫和适应性免疫中都起到重要作用。为了揭示七鳃鳗Myd88的生物学功能, 研究首次从七鳃鳗(Lampetra japonica)中克隆了myd88基因, 其ORF为852 bp, 共编码283个氨基酸, 推测的分子量为32.432 kD, 等电点为6.25, 无信号肽。多重序列比对表明七鳃鳗Myd88的氨基酸序列与其他物种同源性较高, 具有高度保守的N端死亡结构域和C端的TIR结构域的Box1、Box2和Box3基序。实时荧光定量PCR分析表明: myd88基因在七鳃鳗各组织中均有低水平转录表达, 鳃中表达量最高, 其次是肌肉、髓和肾。脂多糖(LPS)体内刺激七鳃鳗后, 七鳃鳗myd88在白细胞中表达量升高最显著, 其次是在鳃中的表达量也明显升高, 表明七鳃鳗Myd88参与七鳃鳗的抗菌免疫过程。此外, LPS刺激七鳃鳗还能诱导TLR信号通路Myd88依赖途径的下游信号分子Irak1、Traf6、Ikkβ和Nfkb在各组织中的转录表达。研究结果表明七鳃鳗中可能存在TLR/Myd88信号通路, 为进一步探究该信号通路参与免疫应答的起源与进化奠定了基础。

THE CLONING AND BIOLOGICAL CHARACTERISTICS OF MYD88 IN LAMPREY AND THE EXPRESSION PATTERN OF ITS DOWNSTREAM PROTEINS

Myeloid differentiation factor 88 (MYD88) is a key adapter protein in Toll-like receptor (TLR) signaling, which plays significant role on the innate and adaptive immunity. In this study, a myd88 gene from lamprey (Lampetra japonica) was obtained. The ORF of myd88 was 852 bp in length, encoding a polypeptide of 283 amino acids. The theoretical molecular weight of lamprey Myd88 was 32.432 kD with a theoretical isoelectric point of 6.25. Lamprey Myd88 was predicted to have no signal peptide. Multiple protein sequence alignment revealed highly conserved death domain in N-terminal, and Box1, Box2, and Box3 in the C-terminal TIR domain, which indicated that lamprey Myd88 was homologous with the MYD88 in other species. Quantitative real-time PCR analysis showed lamprey myd88 gene was extensively expressed in all detected tissues. The highest expression level was observed in gill, which was followed by marrow and kidney. Under in vivo stimulation by lipopolysaccharide (LPS), the expression of myd88 significantly increased in leukocytes, followed by gill, which implicated its role in the defense of L. Japonica against bacteria. Furthermore, increasing expressions of downstream proteins in Myd88-dependent TLR signaling pathway, including Irak1, Traf6, Ikkβ, and Nfkb, were detected in the tested tissues stimulated with LPS. These results suggested that a conserved Myd88-dependent TLR signaling pathway was found in the lamprey, which play a fundamental role on the exploration of the origin and evolution of the signaling pathway in immune response in future.