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系統識別號 U0026-2707201514520000
論文名稱(中文) 探討核仁蛋白在人類A種腸病毒感染中所扮演的角色
論文名稱(英文) The roles of nucleolin in Human enterovirus A infection
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 103
學期 2
出版年 104
研究生(中文) 周韋廷
研究生(英文) Wei-Ting Chou
學號 T36021031
學位類別 碩士
語文別 英文
論文頁數 56頁
口試委員 口試委員-王貞仁
口試委員-余佳益
口試委員-王雅芳
指導教授-張權發
中文關鍵字 人類腸病毒A型  核仁蛋白  克沙奇病毒 
英文關鍵字 Human enterovirus species A  Nucleolin  Coxsackievirus 
學科別分類
中文摘要 人類腸病毒A型(Human enterovirus A, HEV-A)係屬於小RNA病毒科 (Picornaviridae) 、腸病毒屬 (Enterovirus;EV) 之病毒。感染腸病毒後,有的會出現手足口病、疱疹性咽峽炎、無菌性腦膜炎、病毒性腦炎、肢體麻痺症候群等;有的則會造成死亡病例。在HEV-A病毒當中,以腸病毒71型最廣為人知。在我們實驗室先前的研究中,我們利用醣蛋白質體的方法,找到會與EV71有交互作用的醣蛋白-核仁蛋白 (nucleolin, NCL)。我們發現NCL會促進EV71與人類細胞的結合及感染。為了瞭解除了EV71以外,同屬於HEV-A的其他病毒是否同樣會利用NCL來促進感染,本篇研究著重於NCL對其他HEV-A病毒 (如克沙奇病毒A2 (CVA2), CVA4, CVA6, CVA10, CVA16) 感染過程中所扮演的角色。首先我們利用ELISA分析臨床上的5種HEV-A病毒,發現這5種病毒皆會與NCL有交互作用。為了進一步釐清NCL在這些病毒感染中扮演的角色,我們在正常的RD細胞及NCL降低的細胞中感染這5種腸病毒,並透過免疫螢光染色、流式細胞技術、即時PCR、西方墨點法及觀察細胞病變做分析。從結果得知CVA2、CVA6、CVA10在感染細胞過程中會利用NCL做結合對細胞進行感染。然而,NCL的減少對於CVA4及CVA16這2種病毒結合到細胞表面的能力影響較小。此外,我們還發現CVA10產生細胞病變的能力會隨著NCL的表現量下降而減少。本篇研究提出NCL不只會參與EV71的感染,還會參與在其他HEV-A病毒的感染。
英文摘要 Human enterovirus species A (HEV-A) is one of the four species of HEV in the genus Enterovirus in the family Picornaviridae. HEV-A is known to manifest hand-food-mouth diseases in young children and can cause severe neurological diseases such as encephalitis and meningitis. Among HEV-A, enterovirus 71 (EV71) is the most well-known virus. In our previous study, we have identified nucleolin (NCL) as an EV71-interacting glycoproteins by glycoproteomic approaches. NCL plays roles in EV71 binding and infection. In order to understand whether NCL also involves in the binding and infection of other HEV-A viruses, this study aim to investigate the roles of NCL in other HEV-A infection, including coxsackievirus A2 (CVA2), CVA4, CVA6, CVA10, CVA16. Firstly, we analyzed the interaction between NCL with viruses and found that all tested of 5 HEV-A viruses directly interacted with NCL by ELISA. In order to verify whether NCL participates in the infection of these HEV-A viruses, we developed NCL-knockdown cells. We analyzed the binding and infection of the HEV-A viruses to the cells by real-time PCR, immunofluorescence, and flow cytometry. We found that the NCL could affect the binding of CVA2, CVA6, and CVA10 to host cells, and subsequently decrease the viral RNA level in the host cells. However, downregulation of NCL has slightly effects on the binding of CVA4 and CVA16 to the host cells. The reduced cytopathic effect of CVA10 is also accompanied with NCL knockdown in RD cells. These studies showed that NCL not only involved in EV71 infection, but also in other HEV-A viruses infection.
論文目次 Abstract I
中文摘要 II
Acknowledgements III
Index IV
Table Index VI
Figure Index VII
Appendix Index VIII
Abbreviations IX
Introduction 1
Epidemiology 1
Human enterovirus 1
Human enterovirus life cycle 3
Receptors for human enteroviruses 4
Objective, specific aims and experimental design 6
Materials and Methods 7
1. Cell culture and virus propagation 7
2. Determination of virus titers 7
2.1 Plaque assay 7
3. Sucrose gradient purification 8
3.1 PEG precipitation 8
3.2 Virus purification 9
3.3 Virus concentration 9
4. Enterovirus identification 9
5. Interaction between nucleolin and HEV-A virus 10
5.1 EnSpire 10
5.2 Enzyme-Linked Immunosorbent Assay (ELISA) 10
6. Establish nucleolin knockdown cell lines 11
6.1 Lentiviral delivery of short hairpin RNAs 11
6.2 Analysis of cell surface NCL by western blot and flow cytometry 11
7. Virus binding and infection 12
7.1 Immunofluorescence assay 12
7.2 Flow cytometry 13
7.3 Real-time PCR 13
7.4 Immunoblotting 13
7.5 Cytopathic effect (CPE) 14
Results 15
HEV-A serotypes interact with nucleolin 15
NCL knockdown in RD cells decreases HEV-A infection 16
1. Immunofluorescence microscopy 16
2. Flow cytometry 16
3. Real-time PCR 16
4. Immunoblotting and cytopathic effect (CPE) 17
Discussion 18
Conclusion 22
References 23
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