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研究生:洪偉珉
研究生(外文):Wei-Min Hung
論文名稱:腸病毒71型對第一型干擾素的抗性
論文名稱(外文):Resistance of EV71 to Interferon Type I
指導教授:龔思豪
指導教授(外文):Szu-Hao Kung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:腸病毒71型干擾素
外文關鍵詞:EV71IFN
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病毒感染細胞之後會引發細胞產生干擾素反應,其為一種人體內的免疫反應。干擾素分為一~三型,第一型干擾素主要是抗病毒的作用,其誘發的干擾素反應能夠抑制病毒在細胞內進行複製。而某些病毒也會產生抗干擾素的機制。
在本篇研究中,以腸病毒71型做為研究對象,觀察其是否能對第一型干擾素所引發的干擾素反應產生抗性,並且進一步探討其阻斷干擾素反應的作用機制。相較於單純疱疹病毒,腸病毒71型具有抗干擾素作用的效果。接著進一步我們要探討腸病毒71型的何組成物具有抗干擾素作用的機制,首先將腸病毒71型各段基因以轉染的方式送入已經誘發干擾素反應的細胞內,利用雙重冷光酵素報導分析系統 (dual-luciferase reporter assay system) 瞭解干擾素反應下游的訊息傳遞路徑-ISRE (interferon stimulated response element) 活化的程度,發現送入腸病毒2A基因組別的ISRE訊號會有下降的趨勢,表示腸病毒71型的2A蛋白酶有可能是具抗干擾素作用的病毒蛋白質。因此我們以生物資訊網站預測在ISGs (interferon stimulated genes) 中有可能是腸病毒71型2A蛋白酶受質的蛋白質。
本篇研究發現了腸病毒71型抗干擾素作用的現象,並推測其有可能是利用2A蛋白酶進行抗干擾素作用,但究竟是何ISGs被腸病毒71型的2A蛋白酶所影響,還有待日後的研究。
The interferon (IFN) response is an innate immune response in human, and could be induced by viral infection. There are three types of IFNs, type I, II and III, with the type I primarily responsible for antiviral effect. On the other hand, some viruses have evolved anti-IFN mechanisms. In this study, we used enterovirus 71 (EV71) as a model to study whether it could have resistance to the response induced by type I IFN, and further investigated into the possible mechanism. We found that after infection of EV71, there was obvious cytopathic effect (CPE) in cells pretreated with IFN, and the levels of virus yield in the cells were identical between the IFN-untreated and -treated cells. In contrast, there was no discernible CPE in cells infected by herpes simplex virus type 1, an IFN-sensitive control, in the same condition, and the viral yield was reduced to about 50% relative to the IFN-untreated control. The result suggests that EV71 showed certain degree of IFN-I resistance. We then investigated which EV71 components exerted the anti-IFN effect. EV71 gene fragments covering the viral genome were transfected into cells where IFN response had been evoked. We then monitor the IFN–I response by measuring the activity of interferon stimulated response element (ISRE), the downstream promoter of IFN signaling pathway, by the dual-luciferase reporter assay system. We found that EV71 2A gene expression among others caused most significant decrease in the ISRE signal, suggesting that EV71 2Apro maybe at least partly responsible for the anti-IFN response. We further predicted the host interferon stimulated genes (ISGs) that could be the potential substrates of EV71 2Apro by bioinformatics websites. This study revealed the resistance of EV71 to type I IFN, and the effect maybe caused by EV71 2Apro. Which proteins of ISGs were interfered by EV71 2Apro remains to be investigated.
中文摘要 3
Abstract 4
第一章 緒論 5
第一節 干擾素概述 5
第二節 腸病毒概述 10
第三節 單純疱疹病毒概述 13
第四節 研究目的與設計 15
第二章 實驗材料與方法 16
第一節 細胞株的培養與操作 16
第二節 病毒的培養與儲存 18
第三節 測定病毒對干擾素的抗性 20
第四節 干擾素對病毒複製效率的影響 21
第五節 建構帶有腸病毒不同基因片段的表現載體 21
第六節 測定腸病毒不同基因片段對干擾素的影響 29
第七節 雙冷光酵素報導分析系統 30
第八節 腸病毒71型是否會切割IRF5 31
第九節 腸病毒71型是否會切割G6PD 33
第三章 實驗結果 35
第一節 測定腸病毒71型對干擾素的抗性 35
第二節 不同干擾素濃度對腸病毒71型複製效率的影響 35
第三節 測定腸病毒71型不同基因片段對干擾素的影響 36
第四節 IRF5是否為腸病毒71型2A蛋白酶的受質 37
第五節 G6PD是否為腸病毒71型2A蛋白酶的受質 38
第四章 討論 39
第五章 圖表 42
第六章 附錄 50
第七章 參考文獻 56
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