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研究生:江韋緯
研究生(外文):Wei Wei Chiang
論文名稱:日本腦炎病毒在哺乳類動物細胞與蚊子細胞基因重組率之探討
論文名稱(外文):Differential RNA recombination of Japanese encephalitis virus in mammalian cells and mosquito cells
指導教授:陳維鈞陳維鈞引用關係
指導教授(外文):W. J. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:88
中文關鍵詞:日本腦炎病毒基因重組
外文關鍵詞:Japanese encephalitis virusrecombination
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日本腦炎病毒是一種RNA病毒,由於病毒的聚合酶 (polymerase)缺少校正功能,在RNA複製過程中易出錯,突變的累積是造成病毒基因體產生變異,並加速病毒演化的主要原因。然而近年有越來越多的文獻指出病毒基因體的重組現象,也可能影響病毒在宿主中的演化。由於日本腦炎病毒可感染哺乳動物細胞及蚊子細胞,有文獻報導病毒在哺乳動物細胞中易生突變而加速病毒的演化;反之,在蚊子細胞內則呈現減緩的趨勢。類似的情況似乎也出現在病毒的基因重組上,藉由實驗室之前的研究發現日本腦炎病毒株T1P1-S1及CJN-S1可以共同感染哺乳動物細胞(BHK-21)及蚊子細胞(C6/36),然而在這些被病毒共同感染的C6/36細胞中,重組發生率較低於BHK-21細胞,我們更進一步以轉殖外來病毒RNA片段並同時感染Nakayama病毒株觀察重組現象,也發現在BHK-21細胞之重組發生率確實高於C6/36細胞。為了釐清上述現象我們曾以不同比例病毒感染細胞,結果顯示兩病毒之間的競爭與重組發生率並無直接的關係;此外,我們也發現病毒在BHK-21細胞複製速度較C6/36細胞快,當利用低溫培養BHK-21細胞以抑制病毒增殖,可以觀察到病毒複製速度變快,重組發生率也變高;最後,我們觀察到單股RNA在C6/36細胞中降解速度快,反映出RNA穩定度低,我們以pAp提升RNA穩定度,結果顯示在BHK-21細胞中重組發生率隨之提高,但在C6/36細胞中重組發生率卻下降,也許是兩種細胞中內切核醣水解酶的活性不同所造成的影響。結果也顯示病毒複製速度快會提高重組發生率,RNA穩定度低則會降低重組發生率。從以上結果可知,BHK-21細胞中會加速病毒的演化,在C6/36細胞中則反之。


Japanese encephalitis virus (JEV) is a member of Flavivirus that usually causes encephalitis. The genome of JEV contains a positive sense single-stranded RNA, approximatly 11kb in length. Due to the lack of a proof-reading function and error-repairing ability, the mutation of genomic RNA frequently occurs and accumulates during the process of viral evolution. Recently, RNA recombination was found also involved in viral evolution. Naturally, JEV can infect both mammalian and mosquito cells. However, it was reported that the mutation occurs more frequently in mammalian cells, leading to accelerated evolution of the virus. We have recently observed that RNA recombination may also occur in JEV, actually more frequently in mammalian cells (BHK-21). Furthermore, we have transfected a sequent of 5’3’UTR RNA, followed by being infected with Nakayama virus strain to observe the occurrence RNA recombination. The results suggested a similar trend of RNA recombination that occurs more frequently in mammalian cells than in mosquito cells. In spite, it remains unclear how a differential rate of RNA recombination appeared between the two different cell types. To clarify the phenomenon, we first investigated the virus competition by infecting cells with different ratio of two virus strains (T1P1-S1 and CJN-S1); it turns out it is not related to the occurrence of RNA recombination. We subsequently looked at the viral growth rates, showing that the genome of both virus strains replicates more rapid in mammalian cells compared to that in mosquito cells. When infected , mammalian cells were incubated at lower temperature to reduce viral growth, appearing a higher viral growth rate in mammalian cells. Presumably, it may have contributions in increase of RNA recombination. Finally, we measured the possible role of viral RNA stability in the occurrence of RNA recombination. It seems that the single-stranded RNA degraded quicker in mosquito cells than in mammalian cells. We have tried to inhibit 5’-3’exoribonuclease (Xrn1p) by 3’-phosphoadenosine-5’-phosphate (pAp) in order to recover RNA stability in this study. The results showed that RNA recombination rates increased in BHK-21 cells, however, decreased in C6/36 cells. It implicates that RNA stability may be differentially modulated by endoribonuclease in the two different cell lines. In conclusion, a higher rate of viral replication may elevate the RNA recombination rate, on the other hand, the lower RNA stability may reduce it. Taken together, arboviruses accelerate the speed of viral evolution in mammalian cells, otherwise slow down in mosquito cells.
目錄
指導教授推薦書………………………………………………………………………
口試委員會審定書……………………………………………………………………
授權書………………………………………………………………………………iii
誌謝…………………………………………………………………………………v
中文摘要………………………………………………………………………………vi
英文摘要……………………………………………………………………………viii
目錄…………………………………………………………………………………ix
第一章 文獻探討……………………………………………………………………1
第一節 日本腦炎…………………………………………………………………1
一、疾病概述……………………………………………………………………1
二、傳染方式……………………………………………………………………2
三、宿主…………………………………………………………………………2
四、流行病學……………………………………………………………………3
第二節 日本腦炎病毒……………………………………………………………4
一、病毒分類……………………………………………………………………4
二、病毒顆粒構造及基因組成…………………………………………………4
三、病毒轉譯及修飾……………………………………………………………5
四、病毒蛋白質功能……………………………………………………………6
五、病毒生活史…………………………………………………………………6
第三節 黃質病毒的演化…………………………………………………………7
一、病毒演化……………………………………………………………………7
二、RNA病毒基因體重組現象…………………………………………………8
1. 基因重組的分類…………………………………………………………8
2. 基因重組的機制………………………………………………………….10
3. 熱點 (Hot spot)…………………………………………………………10
4.黃質病毒的基因重組………………………………………………………12
三、蚊蟲細胞及哺乳動物細胞在病毒的演化上所扮演的角色…………………13
第二章 研究目的…………………………………………………………………15
第三章 研究設計…………………………………………………………………16
第四章 材料與方法………………………………………………………………17
第一節 材料………………………………………………………………………17
一、細胞株………………………………………………………………………17
二、病毒株………………………………………………………………………17
第二節 實驗方法…………………………………………………………………18
一、細胞繼代培養………………………………………………………………18
二、病毒培養……………………………………………………………………18
三、病毒定量(Plaque assay)……………………………………………………19
四、感染率溶斑試驗(Infectious center assay)…………………………………19
五、分離單一蝕斑 (single plaque isolation)…………………………………20
六、確認T1P1-S1病毒株及CJN-S1病毒株可以共同感染同一個細胞………20
七、細胞RNA的萃取(RNA extraction)及反轉錄反應
(Reversetranscription; RT) …………………………………………21
八、選殖(cloning)…………………………………………………………… 22
九、限制片段長度多態性(RFLP) …………………………………………24
十、定量即時聚合酶反應(Real time RT-PCR) ………………………………25
十一、建構p(+) T1P1-5’3’UTR質體………………………………………26
十二、建構p(+) T1P1-5’3’UTR II及p(-) T1P1-5’3’UTR質體………27
十三、體外轉錄實驗(In vitro transcription assay) ……………………28
十四、轉殖作用(Transfection) ……………………………………………28
十五、以PCR方式偵測轉殖後RNA穩定度……………………………………29
十六、專一性針對重組的RT-PCR……………………………………………30
第五章 結果………………………………………………………………………32
第六章 討論………………………………………………………………………44
第七章 參考文獻…………………………………………………………… ……52
附表…………………………………………………………………………………58
附圖…………………………………………………………………………………62

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