臺灣博碩士論文加值系統

腸病毒71型(EV71) 為小RNA 病毒科(Picornaviridae) 家族成員，在臨床上多造成較輕微的病症，如手足口症；而有時亦會引起較嚴重的疾病，如無菌性腦炎等，致死率高。在先前的研究中指出，人類SCARB2 蛋白是EV 71 的受體之一，並且在EV 71 進入細胞的過程中扮演重要的角色，但是我們對於病毒進入細胞的機制並未完全了解。為了研究是否有其他宿主蛋白會影響EV 71 的感染，在本實驗室先前的免疫沉澱法純化及蛋白質體學的研究中，分析出了許多可能與SCARB2 有交互作用的蛋白。為了進一步分析實際具有交互作用功能的蛋白，我們以人類橫紋肌肉瘤細胞(RD細胞) 作為驗證的實驗模型，利用short hairpin RNA (shRNA) 干擾技術抑制目標基因的表達後，進行EV71 阜陽株螢光表現型(Fy-GFP) 感染，再以實驗室建立的應用篩選平台系統: 流氏細胞儀、即時定量聚合酶連鎖反應、病毒斑實驗，篩選出的目標蛋白CCT chaperonin protein TCP1, CCT2, CCT5 不論是病毒螢光表現、病毒RNA 表現量、病毒力價(titers) 均顯著性下降。默化RD 細胞中TCP1、CCT2、CCT5基因表現後，進行4℃, 40 M.O.I. Fy-GFP 感染實驗結果，再回溫至37℃ 會顯著減少病毒胞吞進入RD 細胞中。建立目標蛋白cDNA 構築，進行免疫沉澱法與免疫染色確認CCT5與SCARB2 蛋白間的交互作用。

Enterovirus (EV71) is a positive single strand RNA virus belonging to the genus Enterovirus in Picornaviridae family. EV71 infection frequently results in hand-foot-and-mouse disease(HFMD), which is characterized by persistent fever, herpangina, and lymphopenia. However, EV71 also leads to a severe form of disease with neurological complications, such as pulmonary edema, aseptic meningitis, encephalitis, paralysis, or even fatality, in children. Human scavenger receptor class B, member 2 (hSCARB2) has been identified as cellular receptors for EV71. hSCARB2 plays critical roles in attachment, viral entry and uncoating, so it is apparent that other factors may be involved in the infection process. We hypothesize that host proteins may interact with SCARB2- EV71 complex to facilitate the infectious process. To identify these proteins, we use a proteomic approach to characterize the proteins co-IP with hSCARB2. One of interacting proteins, TCP1,CCT2, CCT5, appears to play an important role in EV71 infection. Knockdown of TCP1,CCT2, CCT5, efficiently reduces the replication of EV71- GFP- Fuyang strain, revealed by decreased GFP fluorescence, EV71 RNA level, and titer of progeny virus. Using a modified binding assay, we demonstrate that knockdown of TCP1, CCT2, CCT5, expression inhibits the viral internalization/ uncoating steps, but not the viral attachment. Co-IP and immunostain co-localization is performed to confirm the interaction between CCT5 and hSCARB2 in RD cell and Vero cell model. These findings suggest that TCP1, CCT2, CCT5, plays an important role in EV71 infection, especially CCT5.

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誌謝……………………………………………………………………...iii
摘要……………………………………………………………………...iv
Abstract…………………………………………………………………...v
目錄……………………………………………………………………...vi
圖目錄……………………………………………………………….......ix
第一章 序論……………………………………………………..………1
1.1 腸病毒71型…………………………………………………………1
1.2 SCARB2蛋白…………………………………………………….......4
1.3 CCT Chaperonin……………………………………………………...8
第二章 實驗動機…………………………………………....…............11
第三章 材料與方法……………………………………………………12
3.1 實驗材料……………………………………………………..…….12
3.2 實驗方法…………………………………………………....….......19
3.2.1 細胞培養…………………………………………………………19
3.2.2 病毒培養…………………………………………………....……20
3.2.3 以RNA 干擾質體轉染技術製備穩定默化基因細胞………….20
3.2.4 以流式細胞儀偵測默化基因對腸病毒螢光表現型(EV71 Fy-GFP)複製生長的影響………………..……………………………................21
3.2.5以即時定量聚合酶鏈反應偵測默化基因對腸病毒螢光表現型(EV71 Fy-GFP)複製生長的影響…..…………………………………..21
3.2.6 以TRIzol 萃取感染細胞RNA 反轉錄cDNA ………………..22
3.2.7 以病毒斑測試偵測默化基因對腸病毒病毒顆粒(EV71 Fy-GFP)生成影響情形……………………………………………………..……23
3.2.8 以即時定量聚合酶鏈反應偵測默化基因對腸病毒螢光表現型(EV71 Fy-GFP) 低溫病毒結合與病毒內吞的影………………....…..24
3.2.9 製備Human TCP1、CCT2、CCT5 myc-Tag cDNA 構築………..25
3.2.10 轉染…..……………………………………………....…............27
3.2.11 蛋白質定量與西方點墨法……………………………..…........28
3.2.12 以共免疫沉澱法(Co-immunoprecipitaion) 確認蛋白互動性...29
3.2.13 以免疫螢光染色確認CCT5 與SCARB2 的互動……………30
第四章 結果與討論………………………………………...………….31
4.1　以免疫沉澱法及質譜學分析鑑定與EV71 病毒結合受體蛋白SCARB2 過程相關的蛋白質清單…………………………………….31
4.2 基因應用篩選平臺………………………………………………...32
4.2.1 流式細胞儀(Flow Cytometry) 分析…………………………….33
4.2.2即時定量聚合酶鏈反應(quantitative real time-PCR) 分析……..33
4.2.3以病毒斑測試(Plaque Assay) 分析……………………………....34
4.3默化目標基因CCT chaperonin protein: TCP1、 CCT2、 CCT5 對EV71 Fy-GFP結合細胞與胞吞進入細胞的影響……………………..35
4.4以共免疫沉澱法(co-immunoprecipitation) 確認hSCARB2 與目標基因CCT chaperonin protein 的相關性……………………………….36
4.5 以免疫染色法(immunostain) 確認hSCARB2 與目標基因CCT chaperonin protein 共定位……………………………………………..39
參考文獻…………………………………………………………..........40
圖表附錄……..…………………..……………………………………..45

圖目錄
圖一、以免疫沉澱法及質譜學分析鑑定與EV71 病毒結合受體蛋白SCARB2 過程相關的蛋白質清單分析示意圖……………………….45
圖二、基因應用篩選平臺示意圖………………………………………47
圖三、以流式細胞儀偵測穩定默化基因對腸病毒螢光表現型(EV71 Fy-GFP) 複製生長的影響……………………………………………..47
圖四、以即時定量聚合酶鏈反應(quantitative real time-PCR) 偵測穩定默化基因對腸病毒螢光表現型(EV71 Fy-GFP) EGFP RNA與EV71 RNA複製的影響及基因默化效率…………………………………….50
圖五、以病毒斑測試偵測基因默化對腸病毒病毒顆粒生成影響……52
圖六、以即時定量聚合酶鏈反應(quantitative real time-PCR) 偵測穩定默化基因對腸病毒螢光表現型(EV71 Fy-GFP) 結合細胞與胞吞進入細胞的影響……………………………………………………………..53
圖七、在人類橫紋肌肉瘤細胞RD 細胞模型中共免疫沉澱法確認hSCARB2 與CCT chaperonin 的相關性……………………………..55
圖八、在猴腎上皮細胞Vero 細胞模型中共免疫沉澱法確認hSCARB2 與CCT chaperonin 的相關性………………………………………….56
圖九、人類SCARB2 與人類CCT5 共在RD 細胞與Vero 細胞……57

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