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姓名 廖釧妤(Chuan-Yu Liao)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 昆蟲桿狀病毒 IE1 和 IE2 間的交互作用在病毒初始感染的過 程中扮演著關鍵性的角色
(Interactions of IE1 and IE2 Play a Crucial Role for Initiating Baculovirus Infection)
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摘要(中) 昆蟲桿狀病毒被廣泛應用於多種真核生物蛋白的表現工具以及將外來基因運送至細胞中表現的載體。病毒在感染的過程中,基因表現依照時間進行階段性調控,依序被劃分為:早期 (early)、晚期 (late)和極晚期 (very late)。在感染後立即表達的病毒基因在早期的病毒感染過程中起關鍵作用,並影響了病毒之後的基因表達和病毒的繁殖及感染性。研究顯示,昆蟲桿狀病毒立即早期蛋白IE2在昆蟲細胞Sf21及哺乳類細胞Vero E6中是一個很強的轉錄激活蛋白。已知在病毒感染細胞的過程中,宿主的death domain associated protein (Daxx)和Histone H3.3可以與病毒DNA結合以阻斷病毒的入侵,在本實驗室前人的研究中發現IE2會與Daxx及H3.3相互作用以定位病毒DNA。本研究進一步發現在有病毒感染的情況下,IE1會與IE2有共定位現象;若去除病毒將IE1與IE2兩基因共轉染至細胞時,IE2一樣會形成核體 (IE2 nuclear bodies),但是IE1卻是散佈在核中,不會有共定位的現象,推測IE1應該是透過其他病毒因子的幫助與IE2結合。為了找出負責IE1和IE2共定位的病毒基因,我們利用了涵蓋整個134-kb AcMNPV的cosmids 1~7,分別將不同的cosmids與IE1及IE2共轉染至昆蟲細胞,觀察其分佈位置的變化,並發現病毒基因Bgl-II-B片段可能含有影響IE1位置形成並與IE2共定位的基因片段。因此,病毒感染昆蟲細胞時IE2會結合Daxx而定位病毒DNA,進一步幫病毒排除Daxx,接著IE1間接通過IE2與病毒DNA結合,然後IE1會觸發IE2進行自身蛋白降解(self-degradation),以脫離IE2核體的限制,創造適合IE1複製病毒DNA的環境。因此,此研究解析了一個複雜的有關病毒基因產物如何克服寄主免疫系統,定位DNA,以及達成基因表現及DNA複製的初始感染過程。
摘要(英) Baculovirus is widely used as a tool for the expression of eukaryotic proteins and as a carrier for the delivery of foreign genes into cells. The infection cycle can be considered to occur into three stages: early, late, and very late phases. The viral early genes should be responsible for proper viral infections and control subsequent viral gene expression and propagation. The product of the immediately early gene-2 (ie2) is a strong trans-activator for gene expression in insect Sf21 and mammalian Vero E6 cells. It is known that host death domain associated protein (Daxx) and Histone H3.3 can bind and strongly block the function of the intruding viral DNA, unexpectedly, this study found that IE2 could interact with Daxx and H3.3 to locate viral DNA. Further studies showed that IE1 targets IE2 through the help of other viral factors, because IE1 and IE2 do not co-localize by plasmids containing ie1 and ie2 genes, rather co-localization only occurs upon virus infection. In order to identify the viral genes responsible for IE1 and IE2 co-localization, a set of overlapping cosmid clones covering the entire 134-kb AcMNPV genome were co-transfected collectively and separately with IE1 and IE2. We found that the Bgl-II-B fragment of the viral genome may contain the element(s) to affect IE1/IE2 co-localization. Therefore, upon initial baculovirus infection, IE2 can target Daxx to approach viral DNA. After targeting to viral DNA, IE2 helps to remove Daxx, and IE1 in turn targets viral DNA indirectly though IE2. Then IE1 further triggers IE2 self-degradation through ubiquitination pathway to create an environment suitable for DNA replication. Thus, this study delineates a complex story regarding how viral factors overcome host immune restriction, and manage to target their DNA for further gene expression and DNA replication.
關鍵字(中) ★ 桿狀病毒
★ IE1
★ IE2
★ Daxx
★ H3.3
關鍵字(英) ★ Baculovirus
★ IE1
★ IE2
★ Daxx
★ H3.3
論文目次 中文摘要 ………………………………………………………………… i
英文摘要 ………………………………………………………………… ii
誌謝 ………………………………………………………………… iii
目錄 ………………………………………………………………… iv
圖目錄 ………………………………………………………………… vi
一、 緒論………………………………………………………...…. 1
1 -1 昆蟲桿狀病毒簡介……………………………………...……. 1
1 -2 桿狀病毒的應用 …………………………………………..….. 2
1 -3 桿狀病毒基因分類與功能…………………………………… 3
1 -4 病毒早期蛋白 IE1 簡介……………………………………… 3
1 -5 病毒早期蛋白 IE2 簡介……………………………………… 4
1 -6 泛素化………………………………………………………… 5
1 -7 SUMO 轉譯後修飾…………………………………………… 5
1 -8 STUbL (SUMO-targeted ubiquitin ligases) ………………..… 7
1 -9 2-D08……………………………………………..………...…. 7
1 -10 研究動機與目的…………………………………………...…. 7
二、 實驗材料與方法……………………………………………… 9
2-1-1 昆蟲細胞培養………………………………………………… 9
2-1 -2 TC100 配製…………………………………………….….….. 9
2-1 -3 哺乳類細胞培養………………………………………….…... 9
2-2-1 轉染昆蟲細胞……………………………………………..….. 9
2-2-2 轉染哺乳類細胞…………………………………………....… 9
2-3 西方墨點法……………………………………………….…... 10
2-4 RNA 萃取……………………………………………….…….. 10
2-5 mRNA 的逆轉錄……………………………………………… 11
2-6 定量即時聚合?連鎖反應分析……………………………… 11
2-7 免疫螢光染色………………………………………………… 11
2-8-1 聚合?連鎖反應……………………………………………… 12
2-8-2 DNA 瓊脂膠體電泳…………………………………………... 12
2-8-3 DNA 接合作用 …………………………………………….… 13
2-8-4 熱休克法質體轉型…………………………………………… 13
2-8-5 質體純化………………………………………………….…... 13
2-9 Luciferase assay……………………………………………….. 14
v
三、 結果…………………………………………….……………... 15
3-1 IE1 及 IE2 的共定位受到昆蟲桿狀病毒的影響…………….. 15
3-2 觀察其他病毒早期基因對 IE1 及 IE2 共定位現象的影響….. 15
3-3 利用 cosmid 來找尋影響 IE1 及 IE2 共定位的因子………… 16
3-4 IE2 與 IE1 共轉染時, IE2 的表現量和激活能力會下降…… 16
3-5 IE1 並非經由 IE2 上的 IE1 binding motif來調控 IE2 表現… 17
3-6 IE1 是在轉譯層級調控 IE2 的表現………………………… 18
3-7 IE2 通 過 自 身 的 RING domain 來 調 控 自 身 的 降
解……………………………………………………………… 1 8
3-8 利用藥物 2-D08 來阻斷 IE2 的 SUMOylation……………… 19
3-9 SUMOylation 並非是 IE1 調控 IE2 降解的路徑……………. 19
四、 討論…………………………………………………………… 21
五、 參考文獻………..…………………………………………….. 27
六、 圖與表………………..……………………………………….. 31
七、 附錄……………………..…………………………………..… 53
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指導教授 趙裕展 陳盛良(Yu-Chan Chao Shen-Liang Che) 審核日期 2018-7-30
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