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研究生:馮筱庭
研究生(外文):Hsiao-Ting Feng
論文名稱:以奈米半球陣列結構之生醫感測器來檢測日本腦炎病毒與CLEC5A鍵結之關鍵醣基修飾部位
論文名稱(外文):Verifying the Critical Glycol-modification Epitope within the Interaction between Japanese Encephalitis Virus and CLEC5A Via Nano-hemisphere Array Structured Biosensor
指導教授:王國禎
口試委員:張健忠林宜玲
口試日期:2016-06-24
學位類別:碩士
校院名稱:國立中興大學
系所名稱:機械工程學系所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:56
中文關鍵詞:日本腦炎病毒陽極氧化鋁膜電化學阻抗生醫檢測器DC-SIGNCLEC5A
外文關鍵詞:Japanese encephalitis virusAnodic aluminum oxideElectrochemical Impedance SpectroscopyDC-SIGNCLEC5A
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日本腦炎病毒(Japanese encephalitis virus, JEV)會引起腦膜發炎、神經系統和中樞神經損傷,導致長期後遺症,若能提早發現日本腦炎致病機制即可研發疫苗提早預防,減少感染機率。
  日本腦炎病毒外套膜蛋白之醣基修飾會影響病毒感染及複製能力,本研究以陽極氧化鋁膜(Anodic aluminum oxide, AAO)之背阻障層奈米半球陣列結構為電極基板,製作高靈敏度之奈米結構電極,並利用人工突變方式改變日本腦炎病毒外套膜蛋白(D67、N154)上之醣基修飾,再以自組裝單層膜(self-assembly monolayer, SAM)模式 將CLEC5A探針接附於電極表面,最後將不同醣基修飾點之日本腦炎病毒接附於探針,再以電化學阻抗頻譜分析(Electrochemical Impedance Spectroscopy, EIS)量測突變株病毒接附前後電極之阻抗值差異。藉此探討日本腦炎病毒與宿主細胞CLEC5A受器交互作用之關鍵位置。
本研究結果發現JEV野生型病毒皆可與DC-SIGN和CLEC5A作用,但DC-SIGN較CLEC5A有更強之鍵結力。若以不同突變株病毒與CLEC5A反應,則外套膜蛋白胺基酸上第154號沒有修飾N-linked醣基之JEV會導致病毒對CLEC5A之鍵結能力下降,顯示第154號胺基酸上的N-linked醣基修飾影響病毒與CLEC5A之鍵結。


The Japanese encephalitis virus (JEV) can cause meninges inflammation and neuroinflammation, thus leading to lethality. Hence, complete understanding of JEV pathogenesis can benefit the development vaccines to reduce the risk of infection.
The capsid protein of JEV affects its ability of infect ion and replication. In this study, we investigated the pathogenesis of JEV by mutating the protein coat at specific locations (D67 and N154). A extremely sensitive nanostructured biosensor that was built by depositing a gold thin film on the barrier layer surface of an anodic aluminum oxide (AAO) as the electrode was developed for detecting the weak binding between the mutated protein coat and receptors. Receptors CLEC5A and DC-SIGN were attached to different electrodes, respectively, as the probes using the self-assembled monolayer (SAM) method. After immobilizing JEVs with mutated protein coat on the probes, electrochemical impedance spectroscopy (EIS) was than implemented for analyzing the binding locations of JEV on different receptors.
Experimental results illustrate that the wild-type JEV was able to bind to both DC-SIGN and CLEC5A, with a stronger binding affinity to the former. The non-N-linked glycosylated protein coat at the 154th amino acid displayed a lower binding capacity to CLEC5A than the modified version (N-linked glycosylated). The observation indicates that the 154th amino acid on the JEV protein coat heavily influences the JEV-CLEC5A bindings.

Keyword : Japanese encephalitis virus ; Anodic aluminum oxide ; Electrochemical Impedance Spectroscopy ; DC-SIGN ; CLEC5A


中文摘要 i
目錄 vi
圖目錄 ix
第一章 緒論 1
1.1前言 1
1.2黃病毒屬(Flavivirus) 3
1.2.1 日本腦炎病毒(Japanese encephalitis virus,JEV) 5
1.2.2病毒外套膜蛋白之醣基修飾 7
1.3生醫感測器 8
1.3.1 生醫感測器種類及基本原理 9
1.3.2檢測病毒方法之生醫感測器 10
1.4研究動機 12
第二章 實驗原理 14
2.1日本腦炎病毒致病機制 14
2.1.1凝集素與病毒之交互作用 14
2.1.2外套膜蛋白上N-linked醣基化位置與凝集素交互作用之影響 17
2.1.3 生物辨識元件之自組裝結構 19
2.2電化學阻抗頻譜分析(Electrochemical Impedance Spectroscopy, EIS) 20
2.2.1 電化學反應基本原理 20
2.2.2電化學阻抗感測分析原理 21
2.2.3 檢測模擬等效電路元件及檢測圖之關係 22
第三章 實驗材料與方法 24
3.1 實驗材料 24
3.2 實驗設備 26
3.3實驗流程 27
3.4生醫感測器製備 29
3.4.1 奈米半球陣列結構製作 29
3.4.2 高規則奈米半球結構之生醫感測晶片製作 32
3.5 辨識病毒抗原檢體之自組裝結構 35
3.6 電化學阻抗譜檢測 37
第四章 實驗結果與討論 41
4.1 高規則奈米半球結構晶片製作結果 41
4.1.1 奈米半球陣列結構基板之製作結果 41
4.1.2 奈米半球陣列結構晶片之製作結果 42
4.2 探針固定於電極表面結果 42
4.3以電化學阻抗分析檢測JEV與CLEC5A交互關係之檢測 44
4.3.1 JEV外套膜醣基修飾對其與凝集素交互作用之影響 44
4.3.2 JEV野生型與CLEC5A之鍵結 46
4.3.3 JEV外套膜蛋白胺基酸上之N-linked醣基修飾位置對其與DC-SIGN鍵結之影響 47
4.3.4以CLEC5A探討外套膜蛋白胺基酸上之N-linked醣基修飾位置 49
第五章 結論與未來展望 51
5.1結論 51
5.2未來展望 52


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