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研究生:楊玫琳
研究生(外文):Mei-Lin Yang
論文名稱:RNA干擾現象對於抑制腸病毒71型複製能力之研究
論文名稱(外文):Inhibition of enterovirus 71 replication by double-stranded RNA-mediated RNA interference
指導教授:吳文鑾
指導教授(外文):Wen-Luan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:85
中文關鍵詞:腸病毒71型RNA干擾
外文關鍵詞:dsRNARNAiEV71siRNA
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  1998年在台灣暴發腸病毒的大流行,最主要是由腸病毒71型引起,除了手足口病徵之外,有些病例中會引起腦炎、無菌性髓膜炎而造成流行性神經麻痺後遺症,嚴重者會導致死亡,其中嬰兒和小孩為最主要的感染者。有鑑於目前尚無有效疫苗抑制腸病毒71型的感染,因此致力於發展腸病毒疫苗已成為重要目標。RNAi干擾現象是由21-nt siRNAs引起基因沉默的現象,近年來利用RNAi抑制基因表現的機制已在哺乳動物細胞內被廣泛研究。目前已有許多研究指出RNAi可用於哺乳動物細胞對抗病毒的感染上。因此本實驗根據RNAi機制特性,使用長片段dsRNA應用於抑制腸病毒71型的複製。首先利用pol II啟動子轉錄長片段dsRNA,讓RNAi抑制效用比較穩定,因此建構兩個含不同pol II啟動子的載體,分別為(1) pTCY載體:帶有β-actin啟動子可持續於細胞內表現基因,(2) p2-5A載體:含有2'-5'(A)n酵素合成酶基因啟動子,其受干擾素調控基因表現的能力。當腸病毒71型感染細胞時,病毒表現2A蛋白質切酶為切割病毒蛋白之重要切酶,因此選擇2A基因做為本實驗之標的基因。接著,將大小約為450 bp之2A基因片段以正反接方式接入pTCY和p2-5A載體,命名其為pTCY-ds2A及p2-5A-ds2A。將構築好的兩載體分別轉染Vero細胞後,以腸病毒液感染轉染ds2A的Vero細胞,得到抑制病毒能力較高的細胞株Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2。根據細胞病變現象分析,轉染ds2A的細胞株抑制病毒能力高於正常細胞1000倍,且可減少細胞死亡現象。進一步利用RT-PCR檢測腸病毒71型mRNA是否因RNAi作用而減少,以3D基因作為檢測標準,此基因會轉譯為聚合酶,可使腸病毒71型進行複製;根據結果顯示Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2兩細胞株皆減少腸病毒mRNA的複製。另分析細胞凋亡情形,發現轉染ds2A的兩細胞株可減少細胞DNA斷裂。經觀察RNAi效用的持續性結果顯示,Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2細胞株皆可維持RNAi抑制能力11~14天;初步以西方轉漬法分析PKR蛋白質表現,在無病毒感染細胞的情況之下,轉染ds2A的兩細胞株內,PKR蛋白質表現量低而無明顯差別,顯示dsRNA的存在可能不誘導受干擾素調控基因的表現。為了證實細胞抗病毒現象確實與RNAi有關,利用Vero/pTCY-ds2A-2、Vero/p2-5A-ds2A-2和Vero/pTCY-dsE1A進行腸病毒感染實驗,觀察結果Vero/pTCY-dsE1A細胞株大部分細胞死亡和無轉染之Vero細胞並無差異,表示細胞病變具有專一性。綜合以上實驗,利用病毒2A 蛋白切酶基因長片段dsRNA而引起的基因沉默現象,可有效用於抑制腸病毒71型複製。
  In 1998, an epidemic of hand-foot-and-mouth disease caused by enterovirus 71 (EV71) affected thousands of children in Taiwan. In a few cases, more serious symptoms were developed, such as polio-like paralysis, encephalitis, sometimes even resulting in death. Currently, there is no specific treatment or vaccine for this disease. Recently, RNA interference (RNAi) provide an alternative approach with 21-nucleotide small interfering RNA (siRNA) to specifically down-regulate cellular as well as viral gene expression in mammalian cells. In this study, using EV71 infection as a model system, we demonstrated that long double-strand RNA (dsRNA)-mediated RNAi can be effectively used to achieve specific silencing of EV71 replication. First, in order to maintain a better RNAi effect, pol II promoter was used to drive long dsRNA. Two different vectors, pTCY being drived with a β-actin promoter can express gene stably in the cells, and p2-5A being drived with a 2',5'-oligoadenylate synthetase gene promoter can express gene only after interferon induction, were constructed. In addition, previous researches indicated that EV71 2A protease plays an important role in cleavage of EV71 polyprotein. Thus, EV71 2A gene was selected as a target sequence for RNAi. Two dsRNA constructs, pTCY-ds2A and p2-5A-ds2A, containing 450 bp sense and anti-sense fragments of EV71 2A gene were generated. After transfection of pTCY-ds2A and p2-5A-ds2A to Vero cells, two stable clones, Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2, were selected for further investigation. These two transfected cell clones expressing ds2A produced fewer virus particles and the inhibitory effects of virus replication were higher (1000-fold) than those of the non transfected Vero cells. Further experiment of detection of the expression of EV71 3D gene, which encodes viral RNA polymerase for EV71 replication, by RT-PCR showed that both Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2 produced low amount of 3D mRNA. The phenomena of the DNA fragmentation after EV71 infection was also reduced. The duration of EV71-specific RNAi activity in both Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2 were maintained for 11~14 days. Western blotting analysis for detection of PKR expression showed low levels in both transfected cell clones without virus challenge. These observations indicated that long dsRNA could not induce an interferon-stimulated gene, PKR. Furthermore, to confirm that the anti-viral effect was specific from RNAi, Vero/pTCY-ds2A-2,Vero/2-5A-ds2A-2 and Vero/pTCY-dsE1A were used for EV71 infection. The result revealed that the susceptibilities of Vero/pTCY-dsE1A and non transfected Vero cells to EV71-induced cell death were the same. In summary, this study has shown that the inhibition of viral 2A gene expression by long dsRNA-mediated RNA interference might ultimately be useful in treatment of EV71 replication.
中文摘要………………………………………………………………………i
英文摘要………………………………………………………………………ii
致謝..........................................................iv
目錄..........................................................v
表目錄........................................................x
圖目錄................ .......................................xi
附錄目錄......................................................xiii
縮寫字對照表..................................................xiv

第一章 緒論...................................................1
一、RNA干擾機制和生物功能的意義...............................1
二、RNA干擾對於抑制病毒複製的應用.............................3
三、腸病毒的介紹..............................................4
(一)、腸病毒歷史……………………………………………………………4
(二)、腸病毒特性……………………………………………………………5
(三)、腸病毒71型的介紹及相關研究..............................6
四、細胞對干擾素的反應........................................7
五、siRNA和dsRNA對於進行RNAi干擾效用的比較....................8
六、研究目的..................................................9

第二章 材料與方法.............................................11
一、材料......................................................11
(一)、實驗室試劑來源..........................................11
(二)、菌種與質體..............................................11
(三)、細胞株..................................................12
(四)、病毒....................................................12
(五)、細菌培養液..............................................12
(六)、細胞培養液..............................................12
二、引子......................................................12
三、實驗方法..................................................13
(一) 細胞的次培養(cell subculture)............................13
(二) 細胞的計數...............................................13
(三) 病毒的培養及收集.........................................14
(四) RNA的萃取................................................14
(五) 反轉錄聚合酶鏈反應(reverse transcription-PCR)............14
(六) PCR擴增2'-5'(A)n酵素合成酶基因啟動子.....................15
(七) DNA膠體電泳(agarose gel electrophoresis).................16
(八) 由瓊脂凝膠中回收DNA片段..................................16
(九) 去磷酸化.................................................17
(十) T-A Cloning..............................................17
(十一) 接合反應(ligation).....................................17
(十二) 轉型實驗(transformation)...............................18
A. 製備轉型勝任細胞......................................18
B. 大腸桿菌DH5a菌株休克(heat shock)轉型..................18
(十三) 微量製備質體DNA........................................18
(十四) 大量製備質體DNA........................................19
(十五) 限制酵素切割質體(restriction enzyme digestion).........20
(十六) 哺乳動物細胞之質體轉染作用(transfection)...............20
(十七) 哺乳動物細胞株DNA萃取..................................20
(十八) 細胞病變(cytopathic effect, CPE).......................21
(十九) 總蛋白質的萃取.........................................21
(二十) 蛋白質定量分析.........................................22
(二十一) SDS膠片的製備與蛋白質電泳分析........................22
(二十二) 西方轉漬法...........................................23

第三章 結果...................................................25
一、腸病毒71型感染Vero細胞之細胞病變現象(cytopathic effect, CPE)
觀察......................................................25
二、腸病毒71型2A基因的分析與選殖..............................25
三、建構含2-5A (2',5' oligo-adenylate synthetase)基因啟動子之表
現載體....................................................26
四、建構表現腸病毒71型2A基因雙股RNA質體.......................27
五、選殖成功轉染pTCY-ds2A及p2-5A-ds2A載體的Vero細胞株.........28
六、病毒感染轉染ds2A的Vero細胞株..............................29
七、RNAi持續抑制病毒感染能力之觀察............................30
八、Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2細胞株之PKR生成檢測....30
九、抗病毒的專一性實驗........................................31
十、RT-PCR檢測腸病毒3D基因表現量..............................32
十一、腸病毒71型引起細胞凋亡現象(apoptosis)之檢測.............33
十二、轉染ds2A的細胞株在不同時間點下抑制腸病毒71型複製能力之檢測..............................................................33

第四章 討論...................................................35
一、dsRNA標的基因選殖.........................................35
二、腸病毒71型感染轉染ds2A的Vero細胞株........................35
三、兩種不同載體抑制腸病毒71型感染之比較......................36
四、抗病毒的專一性............................................39
五、RNAi對抗病毒感染能力......................................39
第五章 結論與未來展望.........................................41
第六章 參考文獻...............................................43

表目錄

表一、引子對照表..............................................51
表二、用於分析序列的12個腸病毒株..............................52


圖目錄

圖一、實驗基本流程............................................53
圖二、病毒感染前後之Vero細胞型態變化..........................54
圖三、RT-PCR擴增腸病毒71型2A基因片段..........................55
圖四、腸病毒71型2A基因定序結果................................56
圖五、腸病毒71型2A基因序列比對................................57
圖六、yT&A-EV71-2A質體DNA結構圖...............................59
圖七、PCR擴增非洲綠猴腎細胞Vero之2-5A基因啟動子...............60
圖八、p2-5A載體建構流程圖.....................................61
圖九、建構pTCY-ds2A和p2-5A-ds2A質體流程圖.....................62
圖十、pTCY-ds2A及p2-5A-ds2A載體之限制酵素作用檢測.............63
圖十一、PCR檢測細胞DNA是否帶有轉染進入的2A基因................64
圖十二、細胞轉染ds2A後的抗病毒能力測試........................65
圖十三、病毒在細胞內複製能力測試..............................66
圖十四、RNAi抑制病毒複製能力持續效用觀察......................67
圖十五、細胞存活率測試實驗....................................68
圖十六、轉染ds2A細胞株PKR生成測試實驗.........................69
圖十七、RNAi抗病毒專一性實驗..................................70
圖十八、RT-PCR檢測腸病毒3D基因表現量..........................71
圖十九、細胞DNA斷裂試驗.......................................72
圖二十、轉染ds2A的細胞株在不同時間點下抑制腸病毒71型複製能力之檢
測....................................................73


附錄目錄

附錄一、RNAi機制作用過程.....................................74
附錄二、經由dsRNA引起的干擾素反應............................75
附錄三、腸病毒基因結構和複製過程圖...........................76
附錄四、哺乳動物細胞內表現dsRNA的方式........................77
附錄五、12個腸病毒株2A基因序列相似度比較.....................78
附錄六、2-5A酵素合成酶5'端基因序列...........................80
附錄七、yT&A選殖載體譜.......................................81
附錄八、pTCY選殖載體圖譜.....................................82
附錄九、pEGFP-1選質載體圖譜..................................83
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