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研究生:沈育芳
研究生(外文):Yu-Fang Shen
論文名稱:流感病毒核蛋白E339…R416的鹽橋鍵結對流感病毒抑制劑是一個可行的標的
論文名稱(外文):E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors
指導教授:林家立林家立引用關係蔡明道蔡明道引用關係
口試委員:陳玉菁錢偉鈞謝定國
學位類別:博士
校院名稱:國立中興大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:52
中文關鍵詞:流感病毒核蛋白E339…R416的鹽橋鍵結流感病毒抑制劑
外文關鍵詞:influenza virus nucleoproteinE339…R416 salt bridgeinfluenza virus inhibitors
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核醣核酸蛋白質複合體 (ribonucleoprotein complex) 是由聚合酶鹼性蛋白1 (polymerase basic protein 1, PB1)、聚合酶鹼性蛋白2 (polymerase basic protein 2, PB2)、聚合酶酸性蛋白 (polymerase acidic protein, PA) 這三個核醣核酸聚合酶 (RNA-dependent RNA polymerases, RDRP) 的聚合體和核蛋白 (nucleoprotein, NP) 與單股核醣核酸 (ssRNA) 形成的聚合體組成的。核蛋白三聚合體 (trimer) 的晶體結構指出“尾端套環”(tail loop) 及“尾端套環結合”區 (tail loop binding pocket) 的作用對抗病毒藥物的發展是一個有潛力的標的物。近期的兩篇論文證實nucleozin和其衍生物可藉由造成核蛋白聚集進而抑制流感病毒生長。然而這些抑制劑都是經由隨機篩選法 (random screening) 而證實出來的且目前尚不清楚其結合位置及作用機制。我們的研究則是針對另一個機制‘破壞核蛋白與核蛋白之間的交互作用”為目標去找出流感病毒抑制劑。
我們利用合成的“尾端套環” 胜肽片段(胺基酸402到428)可發現其破壞NP的聚合體的聚合現象,在表現“尾端套環” 胜肽片段在細胞培養的實驗中,亦可見其抑制核醣核酸聚合酶的活性和流感病毒的生長。在核醣核酸聚合酶的活性測試的實驗中,我們發現NP的小片段缺失突變Δ402-428和二個點突變R416A和E339A會造成核醣核酸聚合酶活性喪失。但當在細胞內表現NP的點突變R416A和E339A並感染流感病毒,流感病毒的成長會被抑制85%以上。進一步的研究顯現突變的NP與野生型 (wild type, WT) 核蛋白可結合為聚合體,但其有缺陷的結構無法與核醣核酸聚合酶結合。我們亦驗證了二種形式的抑制劑: 合成的胜肽和經由電腦虛擬篩選 (virtual screening) 所篩出的小分子。結果提供了有力的證實: 藉由破壞核蛋白的聚合體的聚合現象可抑制流感病毒的複製。這個研究提供了流感病毒藥物設計的新方向。
The ribonucleoprotein (RNP) molecule consists of the RNA-dependent RNA polymerase (RDRP) polymerase complex (PA, PB1 and PB2) and nucleoprotein (NP) which associates with single-strand RNA to form oligomer. Recently the crystal structure of trimeric NP identified the tail-loop interaction as a potential target site for antiviral development. We tested this hypothesis by showing that the“tail-loop peptide”(residues 402-428) disrupted NP oligomerization in vitro and inhibited RNA-dependent RNA polymerase activities and viral replications in cell cultures. We then showed that Δ402-428 and two site specific mutants R416A and E339A could not substitute for the wild type NP in reconstitution experiments. Importantly, when expressed in cells, R416A and E339A were able to reduce the replication of the H1N1 virus by up to 85%. Further studies suggested that the NP mutants were able to mix with the wild type NP from infecting virus, but the resulting NP oligomers, though with only minor modification structurally, were unable to interact with RDRP. We then tested two types of inhibitors: synthetic peptides and small molecules obtained from virtual screening. The results have provided strong support that disruption of the oligomerization of NP leads to inhibition of the viral replication. This study provides a novel approach to design antiinfluenza drugs.
中文摘要 Ⅰ
英文摘要 Ⅱ
化合物名稱與結構 Ⅲ
表目次 ⅩⅡ
圖目次 ⅩⅢ
第一章 緒論 1
1-1 流感病毒的簡介 1
1-2 流感病毒的結構 2
1-3 流感病毒的核醣核酸聚合酶 3
1-4 流感病毒的核蛋白 4
1-5 抗流感病毒的藥物的發展 7
第二章 材料與方法 9
2-1 抗流感病毒的胜肽與化合物的來源 9
2-2 細胞和流感病毒 10
2-3 質體 10
2-4 冷光酶報導基因分析法 11
2-5 以免疫共沉澱法取得源自流感病毒株的病毒核蛋白的蛋白質電泳樣品 12
2-6 膠體內水解 12
2-7 間接免疫螢光細胞化學法和共軛焦顯微鏡分析野生型和突變型核蛋白的共定位現象 13
2-8 以蛋白質電泳和西方點墨法測試抑制性 14
2-9 以空斑計數試驗測試抑制性 14
2-10 電腦虛擬篩選 15
2-11 引子延伸反應分析 16
2-12 體外轉錄 17
2-13 基因重組的流感病毒的製備 18
2-14 抗病毒力分析 18
2-15 細胞毒性分析 18
2-16 間接免疫螢光細胞化學法 18
第三章 結果與討論 20
3-1 流感病毒的核醣核酸聚合酶聚合體需要核蛋白幫助其功能的進行 20
3-2 流感病毒的核醣核酸聚合酶聚合體與核蛋白互相結合 20
3-3 流感病毒突變型核蛋白E339A、R416A和Δ402–428不能幫助核醣核酸聚合酶聚合體其功能的進行 20
3-4 流感病毒突變型核蛋白E339A和R416A能抑制核醣核酸聚合酶聚合體其功能的進行 21
3-5 流感病毒突變型核蛋白E339A和R416A能抑制流感病毒蛋白質的產生 22
3-6 流感病毒突變型核蛋白E339A和R416A能抑制流感病毒的複製 23
3-7 流感病毒突變型核蛋白E339A和R416A抑制流感病毒複製的機制 23
3-8 以流感病毒的尾端套環為模板設計胜肽抑制劑抑制流感病毒的複製 25
3-9 以電腦虛擬篩選法所篩出的小分子抑制劑抑制流感病毒的複製 26
3-10 化合物3, 7, 12和23與nucleozin和其衍生物的比較 26
第四章 結論 28
參考文獻 48
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