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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張麗冠(Li-Kwan Chang) | |
| dc.contributor.author | Sseu-Pei Hwang | en |
| dc.contributor.author | 黃斯沛 | zh_TW |
| dc.date.accessioned | 2021-06-17T05:05:04Z | - |
| dc.date.available | 2023-08-01 | |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-23 | |
| dc.identifier.citation | Anne-Claude Gingras, Brian Raught, a., Sonenberg, N., 1999. eIF4 Initiation Factors: Effectors of mRNA Recruitment to Ribosomes and Regulators of Translation. Annual Review of Biochemistry 68, 913-963.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71325 | - |
| dc.description.abstract | EB病毒 (Epstein-Barr virus, EBV) 為人類皰疹病毒感染全球約90%的人口，在感染人類之後會將病毒基因轉型至宿主細胞內潛伏，使宿主細胞不朽化。雖然感染EB病毒一般並不會造成疾病，但卻可能引發特定癌症發生。潛伏的病毒受到特定外界環境刺激後會使得病毒重新被再活化 (reactivate)，由潛伏期 (latency) 轉換至溶裂期 (lytic cycle)，製造具有感染性的病毒顆粒。病毒在進行溶裂期複製時首先會表現兩個極早期轉錄因子Rta與Zta，這兩個轉錄因子的能夠進一步開啟病毒其他溶裂期基因表現。表現Rta與Zta的病毒基因BRLF1及BZLF1在病毒基因體上彼此相鄰，能夠由BRLF1基因的啟動子 (Rp) 轉錄出雙基因 (bicistronic) 的BRLF1-BZLF1 mRNA (RZ-mRNA)。過去研究中已經證實Zta蛋白質能夠由RZ-mRNA中被有效的轉譯，而且Rta 蛋白質能夠以cis的方式透過雙基因mRNA中的intercistronic region (ICR) 序列在B淋巴細胞P3HR1細胞株中提升下游BZLF1開放譯讀架的轉譯。本研究發現Rta能夠以trans的方式在293T細胞中活化BZLF1開放譯讀架的轉譯。本研究以建構的雙基因表現質體 (pEGFP-ICR-Luc) 證明Rta為ITAF (IRES trans-acting factor) 能夠反式活化 (trans-activate) 下游基因的表現，且Rta的N端序列為活化時的必要片段。本研究亦對ICR序列進行分段，證明ICR序列中的region I及region II片段對Rta活化雙基因轉譯的重要性。此外本研究也以in vitro RNA-protein pulldown方法證實Rta蛋白質與ICR RNA序列結合。綜合以上結果，本研究證實RZ-mRNA的ICR序列具有IRES活性，而Rta能夠做為ITAF反式活化BRLF1-BZLF1雙基因mRNA中BZLF1開放譯讀區的轉譯，並提升Zta 蛋白質的表現。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus (EBV) is a human herpesvirus, which infects more than 90% of the population. After infection, EBV immortalizes its host cells, establishing latent infection. Although the infection is usually asympotmatic, latent EBV infection is often associated with human cancers. The virus can be reactivated by specific environmental changes and enters the lytic cycle to produce virus particles. At the onset of the lytic cycle, the virus expresses two immediate-early transcription factors, Rta and Zta, which activate EBV’s lytic genes. The genes encoding Rta and Zta, BRLF1 and BZLF1, respectively, are situated adjacent on the viral genome with BRLF1 located upstream. The promoter of BRLF1 (Rp) transcribes a bicistronic BRLF1-BZLF1 mRNA (RZ-mRNA). Earlier study has established that Zta is translated efficiently from the bicistronic mRNA; Rta can elevate the translation of BZLF1 orf located downstream in cis via the intercistronic region (ICR) on the RZ-mRNA in P3HR1 B lymphocytes. This study finds that Rta can trans-activate the BZLF1 translation in 293T cells. By generating a bicistronic reporter plasmid (pEGFP-ICR-Luc), this study verified that Rta acts as an IRES trans-acting factor (ITAF) to activate downstream translation in a trans-acting manner and the N-terminal domain of Rta is essential in the scheme. Deletion analysis of ICR further indicates that the two regions within ICR, region I and region II, are important for the translational activation by Rta. Moreover, RNA-protein pulldown assay confirms that Rta binds to ICR in vitro. Altogether, this study demonstrated that the ICR in RZ-mRNA function as an IRES, while Rta serve as an ITAF that trans-activates the translation of BZLF1 from BRLF1-BZLF1 bicistronic mRNA, thereby promoting the expression of Zta. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T05:05:04Z (GMT). No. of bitstreams: 1 ntu-107-R05B22003-1.pdf: 3139985 bytes, checksum: ce3292c28c7c2cde3a3d7ce3ed256799 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii 目錄 iv 前言 1 1. EB病毒與其生活史 1 2. EB病毒的極早期基因：BRLF1與BZLF1 3 3. 真核細胞的轉譯與轉譯起始 5 4. Cap-independent內轉譯起始 7 5. 病毒的BRLF1-BZLF1雙基因mRNA 9 6. BRLF1-BZLF1雙基因mRNA的intercistronic region與Zta啟動子 10 7. Rta在雙基因RZ-mRNA轉譯中所扮演的角色 13 8. Rta參與細胞轉譯調控所扮演的角色 13 9. 研究動機 14 10. 研究假設 14 材料與方法 15 1. 細胞株 15 2. 質體建構 15 3. 細菌 16 4. 細菌轉型 (transformatin) 16 5. 質體DNA的萃取 (extraction) 17 6. 細胞轉染 (transfection) 17 7. 細胞裂解液 (lysate) 製備 17 8. 雙基因表現系統螢光素酶活性 (luciferase activity) 測試 18 9. In vitro RNA轉錄與萃取 18 10. 以生物素標定RNA 19 11. RNA-protein pulldown assay 19 12. 西方墨點法 (Western blotting) 20 結果 22 1. Rta透過ICR序列反式活化雙基因質體的ZLuc表現 22 2. Rta透過ICR region I與region II序列反式活化雙基因質體的Luc表現 23 3. 缺少N-terminal domain的Rta無法活化下游開放譯讀架轉譯 25 4. 核糖體能夠從ICR序列進入雙基因mRNA開啟下游基因轉譯 25 5. Rta在B細胞中亦能透過ICR序列反式活化雙基因轉譯 26 6. Rta在in vitro條件下會與ICR RNA結合 27 7. Rta的N-terminal domain是與ICR RNA在in vitro結合必要片段 28 討論 30 Rta能夠反式活化雙基因轉譯 31 Rta與RNA結合的活性以及功能區 31 雙基因表現中Rta的角色與重要性 33 ICR具有IRES活性並受到ITAF Rta活化 34 ICR序列的活性區 35 雙基因表現系統所表現之螢光素酶活性來自雙基因mRNA表現 37 Rta與ICR RNA結合 38 Rta參與轉譯調控 40 結論 40 圖表 41 表1、本研究所建構之雙基因表現質體 41 表2、本研究所建構用於in vitro transcription的質體 43 表3、其他本研究所使用之質體 44 表4、本研究所使用之引子序列 46 圖1、EB病毒溶裂期極早期轉錄因子Rta與Zta開啟下游早期以及晚期基因表現 47 圖2、Rta各功能區與特性區示意圖 48 圖3、EB病毒的BRLF1與BZLF1基因的轉錄 49 圖4、雙基因中的ICR序列各片段對於雙基因的轉譯重要性不相同 50 圖5、EB病毒各品系ICR序列比較 51 圖6、雙基因中BRLF1基因帶有缺失突變造成下游開放譯讀架轉譯下降 53 圖7、本研究之假設示意圖 54 圖8-1、pCMV-RZLuc雙基因表現質體與其衍生質體 55 圖8-2、BRLF1的缺失與外源Rta對雙基因系統ZLuc表現的影響 57 圖9、ICR的缺失對於pEGFP-ICR-Luc雙基因表現系統Luc表現的影響 58 圖10、Rta 的N端缺失對反式活化雙基因pEGFP-ICR-Luc的影響 59 圖11、ICR序列前的stem loop對雙基因表現質體Luc表現的影響 61 圖12、Rta在B細胞內對雙基因表現質體Luc表現的影響 62 圖13-1、Rta與ICR RNA的結合 63 圖13-2、Rta與ICR以及ICR 1-85 RNA的結合能力 64 圖14、Rta以及N端缺失的Rta對ICR以及ICR 1-85 RNA的結合能力 65 圖15、透過RNAstructure網站預測ICR全長結構 66 附錄 67 附錄1、EB病毒的生活史 67 附錄2、真核細胞的一般轉譯起始機制以及透過IRES所進行的轉譯起始 68 附錄3、18S rRNA與ICR region I的部分互補關係 70 附錄4、18S rRNA部分序列與結構 71 參考文獻 72 | |
| dc.language.iso | zh-TW | |
| dc.title | Rta促進Epstein-Barr病毒BRLF1-BZLF1雙基因轉譯的機轉 | zh_TW |
| dc.title | Promotion of BZLF1 translation by Rta from the BRLF1-BZLF1 bicistronic mRNA of Epstein-Barr virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉世東(Shih-Tung Liu),張沛鈞(Pey-Jium Chang),羅凱尹(Kai-Yin Lo),廖憶純(Yi-Chun Liao) | |
| dc.subject.keyword | EB病毒,Rta,雙基因mRNA,IRES,ITAF, | zh_TW |
| dc.subject.keyword | Epstein-Barr virus,Rta,bicistronic mRNA,IRES,ITAF, | en |
| dc.relation.page | 82 | |
| dc.identifier.doi | 10.6342/NTU201801775 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-23 | |
| 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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