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研究生:劉書成
研究生(外文):Shu-Cheng Liu
論文名稱:Pyridylimidazolidinones之衍生物對腸病毒七十一型的抗病毒活性
論文名稱(外文):Antiviral activities of pyridyl imidazolidinones against enterovirus 71 variants
指導教授:施信如施信如引用關係
指導教授(外文):Shin-Ru Shih
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:58
中文關鍵詞:腸病毒七十一型
外文關鍵詞:enterovirus 71Pyridyl imidazolidinones
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腸病毒七十一型(EV71)對小孩有高度的感染性,最主要是引起手足口症,但是EV71最讓人覺得害怕的是它會感染人類的神經系統,造成小孩快速的死亡,Pyridyl imidazolidinones為一種具有抗EV71潛力的化合物,經由電腦的模擬被設計出來,BPR0Z-194屬於pyridyl imidazolidinones其中的一種化合物,在我們之前的研究顯示,BPR0Z-194可以抑制腸病毒感染細胞的早期,表示它可以抑制病毒進入細胞內,利用BPR0Z-194去選殖對這個化合物有抗性的病毒,發現這些病毒在VP1裡有共同突變的氨基酸,包括了Arg-22變Gln,Asp-31變Asn,Glu-98 變Lys,Tyr-116變His,Glu-167變Asp,Val-192變Met和Thr-240變Ser。我們利用單點突變的技術在EV71 infectious clone上做出在VP1單點突變的病毒,再以十一個經過化學修飾的pyridyl imidazolidinones作為探針,探討pyridyl imidazolidinones和單點突變病毒之間的交互作用,從化合物敏感性試驗中得知,D31N或E98K這二個點的突變可能會使得VP1疏水區域的開口變得比較大,讓結構比較大的化合物容易進入這一個區域達到抑制病毒的效果,而V192M這一個突變點對於病毒是否對pyridyl imidazolidinones產生抗性扮演著很重要的角色,我們也經由此研究找到幾個可以抑制
EV71VP1-V192M的化合物:D101、D161以及D74。
Enterovirus 71 (EV71) is a highly infectious pathogen which causes hand, foot, and mouth disease (HFMD) in children. EV71 infection is usually accompanying with severe neurological complications and even death. Pyridyl imidazolidinone, discovered by computer-assisted drug design, is a novel class of potent and selective human enterovirus 71 inhibitor. In our previously report, BPR0Z-194, one of the pyridyl imidazolidinones, inhibited virus replication in the early stages, indicating that the compound can inhibit viral adsorption and involves in viral capsid protein function. Using BPR0Z-194 to select and characterize the drug-resistant viruses, we found some common mutations in VP1 region including Arg-22 to Gln, Asp-31 to Asn, Glu-98 to Lys, Tyr-116 to His, Glu-167 to Asp, Val-192 to Met, and Thr-240 to Ser. Here we used site-directed mutagenesis in enterovirus 71 infectious cDNA clone to generate the point mutation viruses. We also used eleven modified pyridyl imidazolidinones as chemical probes to investigate the interactions between compounds and EV71 VP1 protein. From drug susceptibility tests, D31N or E98K mutations in VP1 may let the VP1 hydrophobic pocket which the site of drug interaction become wider, so that more bulky compound can enter this pocket to interfere VP1-receptor binding. We also confirmed that V192M is a key mutation for resistance to the inhibitory effects of the drug. Moreover, from this study, we have identified several modified pyridyl imidazolidinones which is including
D101, D161 and D74 to inhibit the resistant viruses with V192M
mutation.
中文摘要.................................................ii
英文摘要................................................iii
目錄.....................................................iv
圖表目錄.................................................vi
第一章 前言...............................................1
1.1腸病毒的分類與基因結構.................................1
1.2腸病毒七十一型的發現和在台灣的流行.....................1
1.3腸病毒的生活使.........................................2
1.4腸病毒的感染與鞘蛋白質結構之變化.......................3
1.5抗腸病毒七十一型藥物的發展.............................4
1.6研究動機...............................................6
第二章 實驗材料與方法.....................................8
2.1實驗材料...............................................8
2.2實驗方法..............................................10
2.2.1細胞培養............................................10
2.2.2病毒培養............................................10
2.2.3中和試驗以及病毒titer的確認..........................11
2.2.4在含腸病毒七十一型全長基因的infectious clone上作單點突變
.........................................................13
2.2.5基因片段的序列分析..................................14
2.2.6 In vitro RNA transcription...............................14
2.2.7 病毒RNA的轉染試驗.................................15
2.2.8單一週期病毒生長曲線.................................16
第三章 實驗結果..........................................17
3.1 site-direct mutagenesis和序列分析.........................17
3.2 Pyridyl imidazolidinones的化合物和VP1蛋白質的交互作用....17
3.3單一週期病毒生長曲線與real-time RT-PCR.................21
第四章 討論..............................................22
參考文獻.................................................29
表.......................................................33
圖.......................................................42
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