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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 m RNA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c DNA末端快速扩增技术(Rapid amplification of c DNA ends,RACE)获取湖北钉螺MyD88全长c DNA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-q PCR)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c DNA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。q PCR结果显示,检测的所有组织中均有MyD88 m RNA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 m RNA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。
Objective To clone and identify Mycomes 88 (MyD88) gene of Oncomelania hupensis and investigate the expression of MyD88 mRNA in the tissues of Oncomelania hupensis before and after infection with Schistosoma japonicum . Methods The full-length c DNA sequence of MyD88 from Hubei Snail was obtained by rapid amplification of c DNA ends (RACE), and its protein domain was predicted. Multiple sequence alignment and conserved region analysis were carried out to construct phylogenetic tree . Real-time quantitative PCR (RT-qPCR) was used to detect the expression of MyD88 gene in snails before and after infection with Schistosoma japonicum. Results The entire open reading frame (ORF) of MyD88 from Hubei Province was 1 406 bp in length and encoded 468 amino acids. The death domain and the TIR (TIR / TIR) Domain. The similarity to other MyD88 amino acid sequences was 38% -52%. Phylogenetic tree suggested that MyD88 and MyD88 were originated from a common ancestral gene. qPCR results showed that MyD88 m RNA was expressed in all the tissues tested, among which the most abundant was in lymphocytes. Infection with Schistosoma japonicum, MyD88 m RNA in the liver, gonads, blood lymphocytes and other tissues were up-regulated expression, in addition to screw capitellum, up-regulated in the blood lymphocytes in the most significant. CONCLUSION: MyD88 may play an important role in the innate immunity of Oncomelania snails against S. japonicum infection by existence of MyD88-dependent Toll-like receptors (TLRs) signaling pathway.