湖北釘螺髓樣分化因子88的鑒定及其在抗血吸蟲感染中的地位
發(fā)布時間：2018-10-16 14:44
【摘要】：背景髓樣分化因子 88(myeloid differentiation factor 88,MyD88)是連接 Toll樣受體(Toll like receptor,TLR)與蛋白激酶的一種關(guān)鍵蛋白,接受TLR3外所有TLRs的傳導(dǎo)信號,進(jìn)而激活細(xì)胞核內(nèi)多種轉(zhuǎn)錄因子,生成免疫活性物質(zhì),以抵御外來病原體的入侵。MyD88在抗機體抵抗病原體感染過程中發(fā)揮的作用已在多個物種中被證實,然而對軟體類動物湖北釘螺(Oncomelania hupensis)MyD88的研究未見報道。日本血吸蟲感染中間宿主湖北釘螺后,毛蚴與螺體之間與固有免疫相關(guān)的相互作用仍不清楚,研究釘螺固有免疫TLR信號通路中的關(guān)鍵接頭分子MyD88,有助于探索并完善感染早期釘螺體內(nèi)發(fā)生的抗血吸蟲固有免疫防御機制。目的克隆、鑒定湖北釘螺髓樣分化因子88-1(O.HupensisMyD88-1)基因,通過觀察感染日本血吸蟲前后,釘螺各組織中MyD88-1 mRNA表達(dá)水平的變化,探討其在抗血吸蟲感染中的地位。方法在前期獲得部分MyD88-1片段的基礎(chǔ)上,使用cDNA末端快速擴增技術(shù)(Rapid amplification of cDNA ends,RACE)獲取湖北釘螺 MyD88-1 全長 cDNA序列,預(yù)測其蛋白結(jié)構(gòu)以及保守區(qū)域,并與多物種MyD88氨基酸序列進(jìn)行比對,構(gòu)建系統(tǒng)進(jìn)化樹。在RNA水平上,利用實時熒光定量(Real-time quantitative PCR,RT-qPCR)和原位雜交(In Situ Hybridization,ISH)試驗檢測感染血吸蟲前后,釘螺各組織中MyD88-1 mRNA表達(dá)水平的變化。結(jié)果湖北釘螺MyD88-1全長cDNA的開放閱讀框為1406 bp,編碼468個氨基酸,氨基酸序列N端和C端分別存在死亡結(jié)構(gòu)域和TIR結(jié)構(gòu)域。與其他軟體類MyD88氨基酸序列相比,相似性為38%-52%。氨基酸進(jìn)化樹提示,釘螺MyD88-1和光滑雙臍螺MyD88起源于共同的祖先基因。qPCR結(jié)果顯示,在所檢測的釘螺組織中均有MyD88-1 mRNA的表達(dá),于血淋巴細(xì)胞中表達(dá)最為豐富。被日本血吸蟲感染后,除頭足外,釘螺MyD88-1 mRNA在肝臟、生殖腺、血淋巴細(xì)胞中均上調(diào)表達(dá),以血淋巴細(xì)胞中上調(diào)表達(dá)最明顯,高出對照組的4.4×10~4倍。原位雜交試驗檢測顯示,正常螺體MyD88-1 mRNA主要在肝臟、生殖腺的腺體細(xì)胞質(zhì)內(nèi)表達(dá),血吸蟲感染6h、12h、96h后腺體細(xì)胞內(nèi)MyD88-1mRNA表達(dá)增多;正常螺體與感染螺體頭足部肌肉細(xì)胞內(nèi)均未檢測到MyD88-1 mRNA的表達(dá)。結(jié)論湖北釘螺體內(nèi)存在依賴MyD88的TLRs信號通路,該信號通路在血吸蟲感染早期主要通過血淋巴細(xì)胞發(fā)揮抗感染作用。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R532.21
本文編號：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R532.21
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