湖北钉螺髓样分化因子88的鉴定及其在抗血吸虫感染中的地位
发布时间：2018-10-16 14:44
【摘要】：背景髓样分化因子 88(myeloid differentiation factor 88,MyD88)是连接 Toll样受体(Toll like receptor,TLR)与蛋白激酶的一种关键蛋白,接受TLR3外所有TLRs的传导信号,进而激活细胞核内多种转录因子,生成免疫活性物质,以抵御外来病原体的入侵。MyD88在抗机体抵抗病原体感染过程中发挥的作用已在多个物种中被证实,然而对软体类动物湖北钉螺(Oncomelania hupensis)MyD88的研究未见报道。日本血吸虫感染中间宿主湖北钉螺后,毛蚴与螺体之间与固有免疫相关的相互作用仍不清楚,研究钉螺固有免疫TLR信号通路中的关键接头分子MyD88,有助于探索并完善感染早期钉螺体内发生的抗血吸虫固有免疫防御机制。目的克隆、鉴定湖北钉螺髓样分化因子88-1(O.HupensisMyD88-1)基因,通过观察感染日本血吸虫前后,钉螺各组织中MyD88-1 mRNA表达水平的变化,探讨其在抗血吸虫感染中的地位。方法在前期获得部分MyD88-1片段的基础上,使用cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)获取湖北钉螺 MyD88-1 全长 cDNA序列,预测其蛋白结构以及保守区域,并与多物种MyD88氨基酸序列进行比对,构建系统进化树。在RNA水平上,利用实时荧光定量(Real-time quantitative PCR,RT-qPCR)和原位杂交(In Situ Hybridization,ISH)试验检测感染血吸虫前后,钉螺各组织中MyD88-1 mRNA表达水平的变化。结果湖北钉螺MyD88-1全长cDNA的开放阅读框为1406 bp,编码468个氨基酸,氨基酸序列N端和C端分别存在死亡结构域和TIR结构域。与其他软体类MyD88氨基酸序列相比,相似性为38%-52%。氨基酸进化树提示,钉螺MyD88-1和光滑双脐螺MyD88起源于共同的祖先基因。qPCR结果显示,在所检测的钉螺组织中均有MyD88-1 mRNA的表达,于血淋巴细胞中表达最为丰富。被日本血吸虫感染后,除头足外,钉螺MyD88-1 mRNA在肝脏、生殖腺、血淋巴细胞中均上调表达,以血淋巴细胞中上调表达最明显,高出对照组的4.4×10~4倍。原位杂交试验检测显示,正常螺体MyD88-1 mRNA主要在肝脏、生殖腺的腺体细胞质内表达,血吸虫感染6h、12h、96h后腺体细胞内MyD88-1mRNA表达增多;正常螺体与感染螺体头足部肌肉细胞内均未检测到MyD88-1 mRNA的表达。结论湖北钉螺体内存在依赖MyD88的TLRs信号通路,该信号通路在血吸虫感染早期主要通过血淋巴细胞发挥抗感染作用。
[Abstract]:Background MyD88 (myeloid differentiation factor 88 (myeloid differentiation factor 88) is a key protein linking Toll like receptor (Toll like receptor,TLR) with protein kinase. It receives all the transduction signals of TLRs except TLR3, and then activates many transcription factors in the nucleus to produce immunoreactive substances. In order to resist the invasion of foreign pathogens, the role of MyD88 in the process of resisting pathogen infection has been confirmed in many species. However, the study on (Oncomelania hupensis) MyD88 of snail snails in Hubei Province has not been reported. After Schistosoma japonicum infection with Oncomelania hupensis, the interaction between cercariae and snails is still unclear. The study of the key junction molecule MyD88, in the innate immune TLR signaling pathway of Oncomelania hupensis is helpful to explore and improve the innate immune defense mechanism against schistosomiasis in the early stage of infection of Oncomelania hupensis. Objective to clone and identify the myeloid differentiation factor 88-1 (O.HupensisMyD88-1) gene of Oncomelania hupensis and to investigate its role in anti-schistosomiasis infection by observing the changes of MyD88-1 mRNA expression in the tissues of Oncomelania hupensis before and after infection with Schistosoma japonicum. Methods on the basis of partial MyD88-1 fragments obtained, the full-length cDNA sequence of MyD88-1 from Oncomelania hupensis was obtained by cDNA terminal rapid amplification technique (Rapid amplification of cDNA ends,RACE), and its protein structure and conserved region were predicted. The phylogenetic tree was constructed by alignment with MyD88 amino acid sequences of many species. The changes of MyD88-1 mRNA expression in snail tissues before and after schistosomiasis infection were detected by real-time fluorescence quantitative (Real-time quantitative PCR,RT-qPCR) and in situ hybridization (In Situ Hybridization,ISH) tests at RNA level. Results the open reading frame of the full-length cDNA of Oncomelania hupensis MyD88-1 was 1406 bp, encoding 468 amino acids. The N-terminal and C-terminal of the amino acid sequence had death domain and TIR domain respectively. Compared with other software-like MyD88 amino acid sequences, the similarity is 38-52. The phylogenetic tree of amino acids suggested that MyD88-1 and MyD88 of Oncomelania hupensis originated from a common ancestor gene. The results of qPCR showed that MyD88-1 mRNA was expressed in all of the tested snails, and the most abundant in blood lymphocytes. After being infected by Schistosoma japonicum, the expression of MyD88-1 mRNA was up-regulated in liver, gonad and blood lymphocytes except for head and foot, especially in blood lymphocytes, which was 4.4 脳 10 ~ 4 times higher than that in control group. The results of in situ hybridization showed that the expression of MyD88-1 mRNA in normal snails was mainly in the gland cytoplasm of liver and gonad, and the expression of MyD88-1mRNA in glandular cells was increased after 6 h of schistosomiasis infection and 12 h to 96 h of schistosomiasis infection. No expression of MyD88-1 mRNA was detected in the muscle cells of head and foot of normal and infected snails. Conclusion there is a MyD88 dependent TLRs signaling pathway in Oncomelania hupensis, which plays an important role in the early stage of Schistosoma japonicum infection through blood lymphocytes.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.21
本文编号：2274686
[Abstract]:Background MyD88 (myeloid differentiation factor 88 (myeloid differentiation factor 88) is a key protein linking Toll like receptor (Toll like receptor,TLR) with protein kinase. It receives all the transduction signals of TLRs except TLR3, and then activates many transcription factors in the nucleus to produce immunoreactive substances. In order to resist the invasion of foreign pathogens, the role of MyD88 in the process of resisting pathogen infection has been confirmed in many species. However, the study on (Oncomelania hupensis) MyD88 of snail snails in Hubei Province has not been reported. After Schistosoma japonicum infection with Oncomelania hupensis, the interaction between cercariae and snails is still unclear. The study of the key junction molecule MyD88, in the innate immune TLR signaling pathway of Oncomelania hupensis is helpful to explore and improve the innate immune defense mechanism against schistosomiasis in the early stage of infection of Oncomelania hupensis. Objective to clone and identify the myeloid differentiation factor 88-1 (O.HupensisMyD88-1) gene of Oncomelania hupensis and to investigate its role in anti-schistosomiasis infection by observing the changes of MyD88-1 mRNA expression in the tissues of Oncomelania hupensis before and after infection with Schistosoma japonicum. Methods on the basis of partial MyD88-1 fragments obtained, the full-length cDNA sequence of MyD88-1 from Oncomelania hupensis was obtained by cDNA terminal rapid amplification technique (Rapid amplification of cDNA ends,RACE), and its protein structure and conserved region were predicted. The phylogenetic tree was constructed by alignment with MyD88 amino acid sequences of many species. The changes of MyD88-1 mRNA expression in snail tissues before and after schistosomiasis infection were detected by real-time fluorescence quantitative (Real-time quantitative PCR,RT-qPCR) and in situ hybridization (In Situ Hybridization,ISH) tests at RNA level. Results the open reading frame of the full-length cDNA of Oncomelania hupensis MyD88-1 was 1406 bp, encoding 468 amino acids. The N-terminal and C-terminal of the amino acid sequence had death domain and TIR domain respectively. Compared with other software-like MyD88 amino acid sequences, the similarity is 38-52. The phylogenetic tree of amino acids suggested that MyD88-1 and MyD88 of Oncomelania hupensis originated from a common ancestor gene. The results of qPCR showed that MyD88-1 mRNA was expressed in all of the tested snails, and the most abundant in blood lymphocytes. After being infected by Schistosoma japonicum, the expression of MyD88-1 mRNA was up-regulated in liver, gonad and blood lymphocytes except for head and foot, especially in blood lymphocytes, which was 4.4 脳 10 ~ 4 times higher than that in control group. The results of in situ hybridization showed that the expression of MyD88-1 mRNA in normal snails was mainly in the gland cytoplasm of liver and gonad, and the expression of MyD88-1mRNA in glandular cells was increased after 6 h of schistosomiasis infection and 12 h to 96 h of schistosomiasis infection. No expression of MyD88-1 mRNA was detected in the muscle cells of head and foot of normal and infected snails. Conclusion there is a MyD88 dependent TLRs signaling pathway in Oncomelania hupensis, which plays an important role in the early stage of Schistosoma japonicum infection through blood lymphocytes.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.21
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