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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張麗冠 | |
dc.contributor.author | Hsiang-Hung Huang | en |
dc.contributor.author | 黃翔弘 | zh_TW |
dc.date.accessioned | 2021-06-16T17:51:45Z | - |
dc.date.available | 2017-08-27 | |
dc.date.copyright | 2012-08-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-13 | |
dc.identifier.citation | Adams, A. 1987. Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol 61 (5):1743-1746.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64514 | - |
dc.description.abstract | Epstein-Barr virus (EBV)屬於人類皰疹病毒,具有感染上皮細胞及B淋巴細胞的能力,並且是第一個被發現的人類致癌病毒。EBV生活史可分為潛伏期以及溶裂循環,而EBV為了在溶裂循環完成病毒顆粒的複製、組裝及運送會表現出極早期、早期及晚期基因,但目前為止仍有數種蛋白質的功能仍未被仔細研究。然而在EBV中的BBRF2蛋白質,其同源物 (homolog)普遍存在於其他皰疹病毒中,且功能大多與病毒顆粒的晚期功能如組裝與釋放有關,但目前的研究尚未發現EBV的BBRF2蛋白質在其生活史中的功能性。因此本研究首先建立BBRF2的表現系統,純化E. coli中的His-tagged BBRF2以製備抗體,藉此分析其蛋白質特性。然後利用含有EBV的P3HR1細胞以TPA/sodium butyrate誘導病毒引發溶裂循環,以RT-PCR分析BBRF2 mRNA的表現,結果在12小時開始表現,並於之後漸多,約在進入溶裂循環60小時後達到高峰,顯示BBRF2是一種溶裂期的基因。另外透過共軛焦螢光顯微鏡觀察BBRF2在EBV進入溶裂循環後,蛋白質在細胞內的分佈情形,結果顯示,在溶裂循環引發48小時後,BBRF2會與病毒極早期基因Rta在細胞核外有部分重合的現象,在72小時後則會與病毒晚期蛋白質 VCA (Major capsid protein)在細胞核外有部分重合的現象。由於許多病毒的晚期功能被證明與細胞內的各種功能性胞器有密切相關,因此本研究利用融合蛋白GFP-LC3標定細胞內的自噬體 (autophagosome),在48小時透過共軛焦螢光顯微鏡觀察到BBRF2與LC3在細胞核外具有重合的現象,因此推測BBRF2與自噬體的形成可能有關。另外也進一步觀察到選擇性自噬作用 (selective autophagy)中的cargo-protein p62,在細胞自噬作用被誘導後會與BBRF2在細胞質有重合的現象。而在觀察誘導進入溶裂循環的P3HR1細胞中LC3-I轉變成 LC3-II的情形中,發現在EBV的溶裂循環時,LC3-II會增加,表示其自噬作用被誘導。總結以上,BBRF2對EBV在引發溶裂循環後可能扮演重要的角色,未來將更深入探討BBRF2與細胞自噬作用之間的關係。 | zh_TW |
dc.description.abstract | Epstein-Barr virus (EBV) is a human herpesvirus, which infects epithelial cells and B lymphocytes. EBV is the first discovered human carcinoma virus. The life cycle of EBV can be divided into latency and lytic cycle. During the lytic cycle, EBV expresses the immediate-early, early and late genes in order to complete viral replication, viral particles assembly and viral egress. So far, some of EBV proteins’ functions remain unknown. BBRF2 is conserved in all other herpesvirus family, and its in other herpesvirus have been found to involved in virus particle assembly and release. However, the functions of BBRF2 in EBV remain be characterized. In this study, anti-BBRF2 antibody was generated to analyze BBRF2 functions using E. coli expressed His-tagged BBRF2. RT-PCR analysis revealed that the expression of BBRF2 mRNA occurs at 12 hr post induction by TPA/sodium butyrate in P3HR1 cells. The maximum amount of BBRF2 mRNA is at 60 hr after lytic induction, suggesting that BBRF2 is a lytic gene. Moreover, BBRF2 colocalizes with Rta in the cytoplasm as dots at 48 hr after lytic induction by confocal microscopy. At 72 hr post induction, BBRF2 also colocalizes with major capsid protein (VCA) in the cytoplasm. During the late stage of EBV life cycle, some viral proteins interact with different cell organelles in order to complete its life cycle. By using GFP-LC3 as an autophagosome marker, BBRF2 and Rta colocalize with LC3 in the cytoplasm after 48 hours induction, revealing that BBRF2 is related to autophagosome formation. Furthermore, BBRF2 colocalizes with p62, a cargo protein in selective autophagy, in the cytoplasm after starvation. Finally, this study found LC3-I is converted into LC3-II during lytic induction, suggesting that lytic cycle induces autophagy. Taken together, BBRF2 plays a crucial role in EBV lytic activation. The relationship between BBRF2 and autophagy will be elucidated in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:51:45Z (GMT). No. of bitstreams: 1 ntu-101-R99b22028-1.pdf: 6941364 bytes, checksum: e7c1ea8bd77b126403a56b3c71978a86 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 中文摘要 ii
Abstract iv 目錄 vi 前言 1 一、 Epstein-Barr virus (EBV) 1 二、 EBV的遺傳物質與結構特性 1 三、 EBV的致病性與相關疾病 2 四、 EBV的生活史 2 EBV的潛伏期 3 EBV的溶裂循環 3 EBV的組裝與釋放 4 五、 EBV的間質蛋白質 (EBV tegument protein) 5 六、 EBV的未知蛋白質BBRF2 6 七、 自噬作用 (Autophagy) 7 八、 自噬體與病毒的相關研究 8 自噬作用與細胞免疫反應 8 自噬作用與病毒的交互作用 8 自噬作用與皰疹病毒的相關研究 9 研究目的 11 材料與方法 12 一、 菌種與細胞株 12 二、 質體與抗體 12 三、 大腸桿菌轉型作用 12 四、 質體DNA的萃取 12 五、 細胞轉染 (Transfection) 13 六、 EBV溶裂循環誘導 13 七、 免疫螢光染色分析 (Immunofluorescence analysis) 13 八、 免疫沉澱法 (Immunoprecipitation, IP) 14 九、 西方點墨法分析 (Western blot) 14 十、 蛋白質誘導表現 15 十一、 變性蛋白質純化 (Ni-NTA denature protein purification) 15 十二、 SDS-PAGE膠體蛋白質染色分析 16 十三、 抗體製備 16 十四、 細胞starvation處理 16 結果 17 一、 BBRF2在溶裂期的表現時間 17 二、 BBRF2抗體的製備 17 三、 BBRF2在EBV進入溶裂循環時之分布位置 18 四、 BBRF2與Rta和細胞內參與自噬作用之蛋白質LC3的交互作用 19 五、 BBRF2與p62的交互作用 19 六、 BBRF2與EBV major capsid protein (VCA)的交互作用 19 七、 自噬作用對EBV 溶裂期之影響 20 討論 21 圖表 27 表一、本研究使用之質體 27 表二、本研究使用之抗體 29 附圖 30 圖1、EBV的生活史 30 圖2、以RT-PCR分析BBRF2 mRNA在溶裂循環不同時間點的表現量 32 圖3、His-BBRF2的表現與純化 33 圖4、GFP-BBRF2與Rta在P3HR1細胞引發EBV進入溶裂期時的表現位置 34 圖5、GFP-BBRF2與Rta在P3HR1細胞引發EBV進入溶裂期時的表現位置 35 圖6、GFP-BBRF2與Rta在NA細胞引發EBV進入溶裂期時的表現位置 36 圖7、GFP-BBRF2與Rta-RFP在293T細胞中的分布情形 37 圖8、Flag-BBRF2與GFP-LC3在P3HR1細胞誘導EBV進入溶裂循環後的分布情形 38 圖9、Rta與GFP-LC3在P3HR1細胞誘導EBV進入溶裂循環後不同時間點中的分布情形 39 圖10、GFP-BBRF2與RFP-p62在293T細胞經starvation處理後的免疫螢光染色分析 40 圖11、GFP-BBRF2與VCA在EBV溶裂期的分佈情形 41 圖12 、Flag-BBRF2與HA-VCA在細胞內結合 42 圖13、LC3在P3HR1細胞引發EBV進入溶裂循環後的表現 43 附錄 44 附錄1、HSV-1病毒顆粒釋放路徑 (節錄自: Nat Rev Microbiol. 2011 May; 9(5):382-94) 44 附錄2、Autophagosome的形成機制 (節錄自: Annu. Rev. Immunol. 2009. 27:423–49) 45 附錄4、病毒與autophagy除了免疫系統外的交互作用 (節錄自: T.X. Jordan, G. Randall / Microbes and Infection xx (2011) 1e14) 48 附錄5、Herpesviridae與autophagy間的交互作用 (節錄自Taylor et al. Herpesviridae 2011, 2:2) 49 參考資料 50 | |
dc.language.iso | zh-TW | |
dc.title | Epstein-Barr virus BBRF2蛋白質的功能性分析 | zh_TW |
dc.title | Functional analysis of Epstein-Barr virus BBRF2 protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉世東,張沛鈞,張世宗 | |
dc.subject.keyword | EBV,間質蛋白質,晚期基因,自噬作用, | zh_TW |
dc.subject.keyword | EBV,tegument proteins,late gene,autophagy, | en |
dc.relation.page | 69 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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