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系統識別號 U0026-2306201012032900
論文名稱(中文) 研究細胞表面醣分子在腸病毒71型感染中扮演之角色
論文名稱(英文) Characterize the Role of Cell Surface Glycans on the Infection of Enterovirus 71
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 98
學期 2
出版年 99
研究生(中文) 劉玥彤
研究生(英文) Yueh-Tung Liu
學號 T3697407
學位類別 碩士
語文別 中文
論文頁數 93頁
口試委員 指導教授-張權發
口試委員-王貞仁
口試委員-余俊強
中文關鍵字 腸病毒71型  唾液酸  唾液酸轉移酶 
英文關鍵字 enterovirus 71  sialic acid  sialyltransferase 
學科別分類
中文摘要 腸病毒71型(EV71)屬於小RNA病毒科(Picornavirade),於臨床上常導致五歲以下幼兒的手足口病,並且伴隨著嚴重的神經性併發症。然而,對於腸病毒71型的細胞附著、細胞入侵以及其感染機制至今尚未完全清楚。根據兩篇發表在Nature Medicine的文獻指出,依照實驗細胞株及病毒株的差異,腸病毒71型可藉由辨認細胞表面human scavenger receptor class B, member 2以及human P-selectin glycoprotein ligand-1作為其感染受體。作者亦發現當細胞經由單株抗體作用後,卻不能完全抑制腸病毒71型的感染。因此,作者認為在腸病毒71型感染細胞的過程中,應存在有multiple receptors參與病毒的致病機轉。目前已知細胞表面的醣類分子參與在許多病毒感染細胞的過程中,包括:流行性感冒病毒、輪狀病毒與腸病毒70型…等。因此,本論文想要探討細胞表面之醣類分子在腸病毒71型附著與感染細胞的過程中所扮演的角色與重要性。首先,我們表現及純化出具有酵素活性之α2,6-與α2,3-唾液酸轉移酶,並且透過化學酵素學的方式合成出8個末端帶有唾液酸之聚丙烯醯-寡醣受質。接著,利用蔗糖濃度梯度大量純化出具有感染能力之小鼠適應株MP4病毒顆粒,並透過醣微矩陣的分析,觀察病毒與醣類分子的結合反應。結果顯示腸病毒71型與sialyl Lewis x有較高的結合強度。為了更進一步觀察細胞表面之N-linked、O-linked與末端帶有唾液酸之醣鏈在腸病毒71型致病機轉中所扮演的角色,我們將RD細胞分別經由tunicamycin、benzyl-α-GalNAc或neuraminidase處理,並分別利用酵素免疫分析法、流式細胞儀與即時定量聚合酶連鎖反應分析法測量腸病毒71型的結合能力與感染能力。結果顯示,在病毒感染細胞的過程中,腸病毒71型會與細胞表面末端帶有唾液酸之醣類分子進行結合,進而增加病毒結合與感染細胞的能力。除此之外,我們將細胞經由兩種唾液酸結合凝集素處理,發現凝集素具有抑制病毒感染細胞的能力。總結,我們認為RD細胞表面末端帶有唾液酸之醣類分子,確實參與在腸病毒71型致病機轉中,並扮演相當重要的角色。
英文摘要 Enterovirus 71 (EV71), an unusual human pathogen of the family Picornaviridae, is the major agent cause of hand-foot-and-mouth disease (HFMD) associated with severe neurological disease. The mechanisms of virus attachment, invasion and virulence determinants of EV71 are still unclear. Recent studies showed that EV71 recognizes not only scavenger receptor B2 (SCARB2) but also human P-selectin glycoprotein ligand-1 as cellular receptors depended on difference cell types or virus strains. However, the infection of EV71 could not be inhibited by specific antibody entirely, suggesting that these might be other pathway or receptor for virus infection during early stage. Because cell surface glycans have been demonstrated to involve in the infection of viruses including influenza virus, adenovirus, and enterovirus 70, we want to investigate the role of cell surface glycans during the infection of EV71. First, we expressed α2,3- and α2,6-sialyltransferases and subjected the enzymes for producing eight sialylated glycans. Then, we analyzed the carbohydrate binding property of purified the fourth passage of mouse-adapted EV71 strain (MP4) by glycan microarray. The glycan binding results indicated that cell surface sialyl Lewis x (sLex) might play important roles on mechanisms of virus infection. In order to explore the significance of cell surface N-linked, O-linked, and sialylated glycans during EV71 binding and infection, we had treated RD cells with tunicamycin, benzyl-α-GalNAc or neuraminidase and quantified the bound of amplified virus by ELISA assay, flow cytometry, real-time PCR, and fluorescence microscopy. The results showed that the cell surface sialic acid containing biomolecules were essential for EV71 binding and infection. In addition, interactions between virus and RD cells could be inhibited by Sambucus nigra lectin (SNA, NeuAcα2-6 glycan binding lectin) and Maackia amurensis lectin (MAA, NeuAcα2-3 glycan binding lectin) in dose dependent manner. In conclusion, sialylated glycans on RD cell surface played a critical role during EV71 binding and infection.
論文目次 Abstract I
中文摘要 II
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 X
縮寫檢索表 XI
第一章 研究背景 1
1-1 醣化的功能與生理意義 1
1-2 唾液酸(sialic acid)之生理特性 2
1-3 醣鏈修飾技術與醣微矩陣(glycan microarray) 2
1-4 腸病毒71型之特性 3
1-5 腸病毒71型之臨床症狀與致病機轉 5
1-6 病毒與醣類分子之交互作用 6
第二章 研究目標 8
第三章 實驗材料與方法 9
3-1 利用α2,6-sialyltransferase與α2,3-sialyltransferase擴大glycan library 9
3-1-1 重組蛋白的表現及純化 9
3-1-2 重組蛋白SDS-PAGE膠體電泳 10
3-1-3 重組蛋白的西方點墨法分析 10
3-1-4 利用高效能液相層析儀系統(HPLC)偵測重組蛋白之活性 11
3-1-5 利用唾液酸轉移酶進行醣鏈合成反應 12
3-1-6 利用AlphaScreen偵測α2,6-sialyltransferase醣鏈合成反應之效能 12
3-1-7 利用AlphaScreen偵測α2,3-sialyltransferase醣鏈合成反應之效能 13
3-2 細胞培養與病毒純化 13
3-2-1 培養人類橫紋胚胎肌肉瘤細胞(human rhabdomyosarcoma cell:RD cell) 13
3-2-2 腸病毒71型小鼠適應株製備 14
3-2-3 利用PEG/NaCl沉澱腸病毒71型 14
3-2-4 蔗糖濃度梯度(Sucrose gradient)反應 15
3-2-5 重組第四代腸病毒71型小鼠適應株之線型質體(linear plasmid)純化技術 16
3-2-6 細胞體外轉錄技術(In vitro RNA transcription) 17
3-2-7 病毒RNA轉染(Transfection)技術 18
3-2-8 病毒效價測試(溶菌斑試驗) 19
3-3 老鼠EV71 1gG3單株抗體製作 20
3-3-1 融合瘤細胞(Hybridoma)之培養 20
3-3-2 單株抗體的製備 20
3-3-3 單株抗體的純化 20
3-3-4 單株抗體效價測試 21
3-4 即時定量聚合酶連鎖反應(Real-time PCR) 22
3-4-1 利用病毒RNA配置RT-PCR之標準曲線 22
3-4-2 病毒RNA之萃取技術 23
3-5 利用AlphaScreen偵測醣類分子與腸病毒71型之結合反應 24
3-6 利用流式細胞儀與螢光顯微鏡分析RD細胞表面醣類結構 25
3-6-1 利用流式細胞儀做醣類抗體分析 25
3-6-2 利用流式細胞儀做凝集素分析 26
3-6-3 利用免疫螢光染色法做細胞醣類結構分析 26
3-7 細胞N-linked glycan與O-linked glycan在EV71感染RD細胞過程之功能分析 27
3-7-1 利用酵素免疫分析法(ELISA)測試EV71結合RD細胞之能力 27
3-7-2 利用流式細胞儀測試EV71結合RD細胞之能力 27
3-7-3 利用real-time PCR測試EV71結合與感染RD細胞之能力 28
3-7-4 利用EGFP reporter system測試EV71感染RD細胞之能力 29
3-8 細胞表面Sialic acid在EV71感染RD細胞過程之功能分析 29
3-8-1 利用酵素免疫分析法測試細胞之Sialic acid影響病毒結合細胞之能力 29
3-8-2 利用流式細胞儀測試細胞之Sialic acid影響病毒結合細胞之能力 30
3-8-3 利用real-time PCR測試細胞之Sialic acid影響病毒結合與感染細胞之能力 30
3-8-4 利用EGFP reporter system測試Sialic acid影響病毒感染細胞之能力 31
3-9 利用凝集素抑制腸病毒71型感染RD細胞之實驗設計 31
3-9-1 利用酵素免疫分析法(ELISA) 觀察凝集素抑制EV71結合細胞之能力 31
3-9-2 利用real-time PCR觀察凝集素抑制EV71結合細胞之能力 32
3-9-3 利用EGFP reporter system觀察凝集素抑制EV71感染細胞之能力 32
3-9-4 利用real-time PCR觀察凝集素抑制EV71感染細胞之能力 33
3-10 統計方法 33
第四章 結果 34
4-1 利用α2,6-sialyltransferase與α2,3-sialyltransferase擴大glycan library 34
4-1-1 利用α2,6-sialyltransferase重組蛋白進行醣鏈末端合成反應 34
4-1-2 利用α2,3-sialyltransferase重組蛋白進行醣鏈末端合成反應 35
4-2 評估腸病毒71型之純化效率 35
4-3 老鼠EV71 1gG3單株抗體效能分析 36
4-4 即時定量聚合酶連鎖反應(Real-time PCR)標準曲線之製作 36
4-5 利用AlphaScreen分析醣類分子與腸病毒71型之結合反應 37
4-6 利用流式細胞儀與螢光顯微鏡分析RD細胞表面之醣類結構 38
4-7 分析細胞N-linked glycan與O-linked glycan在EV71感染RD細胞過程之角色 39
4-7-1 細胞N-linked glycan與O-linked glycan對EV71結合細胞之能力的影響 39
4-7-2 細胞N-linked glycan與O-linked glycan對EV71感染細胞之能力的影響 40
4-8 分析細胞表面唾液酸(sialic acid)在腸病毒71型感染RD細胞過程之角色 41
4-8-1 細胞表面Sialic acid對EV71結合細胞之能力的影響 41
4-8-2 細胞表面Sialic acid對EV71感染細胞之能力的影響 42
4-9 評估凝集素保護細胞抵抗腸病毒71型感染之能力 43
4-9-1 凝集素抑制EV71結合RD細胞 43
4-9-2 凝集素抑制EV71感染RD細胞 44
第五章 討論 46
結論 52
參考文獻 53
圖 59
表 84
附錄 88

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