Objective: To identify a myeloid differentiation factor 88 (MyD88) in Oncomelania hupensis, and characterize the role of MyD88 against Schistosoma japonicum infection.
Methods: The complete cDNA of MyD88 in O. hupensis was obtained by using rapid amplification of cDNA ends (RACE), and homologues sequences and conserved domains were aligned and the structure of MyD88 was predicted either. A phylogenetic tree of MyD88 was further constructed with other species. In addition, the mRNA expression level of O. hupensis MyD88 before and after S. japonicum infection was investigated by real-time quantitative PCR (RT-qPCR).
Results: The cDNA of O. hupensis MyD88 consisted of 1 406 bp open reading frame (ORF), encoding 468 amino acid residues, which contained death domain and Toll/interlrukin-1 receptor (TIR) domain, the typical features of MyD88 family proteins. The predicted amino acid sequence of O. hupensis MyD88 shared 38%-52% identity with other mollusc. O. hupensis MyD88 was phylogenetically closeted to Biomphalaria glabrata MyD88. The O. hupensis MyD88 existed in all selected tissues and expressed highly in hemocyte, up-regulated after S. japonicum infection in all selected tissues except cephalopodium, especially higher in whole snail and hemocyte.
Conclusions: MyD88-dependent signaling pathway is present in O. hupensis and plays an important role in innate immune response against S. japonicum infection.
［摘要］目的 克隆、鉴定湖北钉螺 (Oncomelania hupensis) 髓样分化因子88 (MyD88) 基因, 并通过观察感染日本血吸虫 前后钉螺各组织中MyD88 mRNA表达水平的变化, 探讨其在抗血吸虫感染固有免疫中的地位。方法 通过cDNA末端 快速扩增技术 (Rapid amplification of cDNA ends, RACE) 获取湖北钉螺MyD88全长cDNA序列, 预测其蛋白结构域, 并进 行多序列比对及保守区域分析, 构建系统进化树。使用实时荧光定量PCR (Real-time quantitative PCR, RT-qPCR) 技术检 测MyD88基因在血吸虫感染前后钉螺各组织中的表达变化。结果 湖北钉螺MyD88全长cDNA开放阅读框 (Open reading frame, ORF) 1 406 bp, 编码468个氨基酸, 蛋白N端和C端分别存在死亡结构域和TIR (Toll/interlrukin-1 receptor, TIR) 结构域。与其他软体类MyD88氨基酸序列比对, 相似性为38%~52%; 系统进化树提示钉螺MyD88和光滑双脐螺MyD88 起源于共同的祖先基因。qPCR结果显示, 检测的所有组织中均有MyD88 mRNA的表达, 其中血淋巴细胞中表达最为丰 富。感染日本血吸虫后, 除钉螺头足外, MyD88 mRNA在肝脏、生殖腺、血淋巴细胞等组织中均有上调表达, 以血淋巴细 胞中上调表达最显著。结论 湖北钉螺存在依赖MyD88的TLRs (Toll-like receptors, TLRs) 信号通路, MyD88分子可能在 钉螺对抗日本血吸虫感染的固有免疫中发挥重要作用。.
Keywords: Innate immunity; Myeloid differentiation factor 88 (MyD88); Oncomelania hupensis; Schistosoma japonicum; Toll-like receptor signaling pathway.