Skip navigation

DSpace

機構典藏 DSpace 系統致力於保存各式數位資料(如:文字、圖片、PDF)並使其易於取用。

點此認識 DSpace
DSpace logo
English
中文
  • 瀏覽論文
    • 校院系所
    • 出版年
    • 作者
    • 標題
    • 關鍵字
  • 搜尋 TDR
  • 授權 Q&A
    • 我的頁面
    • 接受 E-mail 通知
    • 編輯個人資料
  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 微生物學科所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49518
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor張鑫(Shin C. Chang)
dc.contributor.authorWei-Chen Shinen
dc.contributor.author辛惟臻zh_TW
dc.date.accessioned2021-06-15T11:32:36Z-
dc.date.available2021-08-26
dc.date.copyright2016-08-26
dc.date.issued2016
dc.date.submitted2016-08-17
dc.identifier.citation1. Coleman CM, Frieman MB. 2013. Emergence of the Middle East Respiratory Syndrome Coronavirus. PLoS Pathog 9:e1003595.
2. deGroot RJ, Baker SC, BaricRS, BrownCS, Drosten C, EnjuanesL, RA F, GalianoM, Gorbalenya AE, Memish ZA, PerlmanS, Poon L, Snijder EJ, Stephens G, Woo PC, Zaki AM, Zambon M, Ziebuhr J. 2013. Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J Virol 87:7790-7792.
3. Raj VS, Mou H, Smits SL, Dekkers D H, Müller MA, Dijkman R, Muth D, Demmers J A, Zaki A, Fouchier R A, Thiel V, Drosten C, Rottier P J, Osterhaus A D, Bosch B J, Haagmans B L. 2013. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495:251-254.
4. Lau SK, Li KS, Tsang AK, Lam CS, Ahmed S, Chen H, Chan KH, Woo PC, Yuen KY. 2013. Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus. J Virol 87:8638-8650.
5. Wang Q, Qi J, Yuan Y, Xuan Y, Han P, Wan Y, Ji W, Li Y, Wu Y, Wang J, Iwamoto A, Woo PC, Yuen KY, Yan J, Lu G, Gao GF. 2014. Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26. Cell Host Microbe 16:328-337.
6. Yang Y, Du L, Liu C, Wang L, Ma C, Tang J, Baric RS, Jiang S, Li F. 2014. Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc Natl Acad Sci U S A 111:12516-12521.
7. Durai P BM, Shah M, Choi S. 2015. Middle East respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control. Exp Mol Med 47:e181.
8. Shehata MM, Gomaa MR, Ali MA, Kayali G. 2016. Middle East respiratory syndrome coronavirus: a comprehensive review. Front Med 10:120-136.
9. Vennema H, Godeke GJ, Rossen JW, Voorhout WF, Horzinek MC, Opstelten DJ, Rottier PJ. 1996 Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J 15:2020-2028.
10. Walls AC, Tortorici MA, Bosch BJ, Frenz B, Rottier PJ, DiMaio F, Rey FA, Veesler D. 2016. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. Nature 531:114-117.
11. Millet JK, Whittaker GR. 2014. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. Proc Natl Acad Sci U S A 111:15214-15219.
12. Boscarino JA, Logan HL, Lacny JJ, Gallagher TM. 2008. Envelope protein palmitoylations are crucial for murine coronavirus assembly. J Virol 82:2989-2999.
13. de Haan CA, Vennema H, RottierP J. 2000. Assembly of the coronavirus envelope: homotypic interactions between the M proteins. J Virol 74:4967-4978.
14. Chang CK, Lo S C, Wang YS, Hou MH. 2016 Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein. Drug Discov Today 21:562-572.
15. Kuo L, Hurst-Hess KR, Koetzner CA, Masters PS. 2016 Analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras. J Virol 90 4357-4368
16. Verma S, Bednar V, Blount A, Hogue BG. 2006 Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein. J Virol 80:4344-4355
17. Narayanan K, Chen C J, Maeda J, Makino S. 2003. Nucleocapsid-independent specific viral RNA packaging via viral envelope protein and viral RNA signal. J Virol 77:2922-2927.
18. Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, Snijder EJ. 2008. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6:e226. .
19. Yang Y, Zhang L, Geng H, Deng Y, Huang B, Guo Y, Zhao Z, Tan W. 2013. The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein Cell 4:951-961.
20. Pasternak AO, Spaan WJ, Snijder EJ. 2006. Nidovirus transcription: how to make sense...? J Gen Virol 87:1403-1421.
21. Stadler K, Masignani V, Eickmann M, Becker S, Abrignani S, Klenk HD, Rappuoli R. 2003. SARS--beginning to understand a new virus. Nat Rev Microbiol 1:209-218
22. Lai MM, Patton CD, Stohlman SA. 1982 Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length. J Virol 44:487-492.
23. Lai MM, Patton CD, Stohlman SA. 1982. Further characterization of mRNA's of mouse hepatitis virus: presence of common 5'-end nucleotides. J Virol 41:557-565.
24. Sawicki SG, Sawicki DL. 1990 Coronavirus Transcription: Subgenomic Mouse Hepatitis Virus Replicative Intermediates Function in RNA Synthesis. J Virol 64:1050-1056.
25. Sethna P, Hung S, Brian D. 1989. Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci U S A 86:5626-5630.
26. Wu CH, Chen PJ, Yeh SH. 2014. Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription. Cell Host & Microbe 16 462–472.
27. Hsieh PK, Chang SC, Huang CC, Lee TT, Hsiao CW, Kou YH, Chen IY, Chang CK, Huang TH, Chang MF. 2005. Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent. J Virol 79:13848-13855.
28. Qin L, Xiong B, Luo C, Guo ZM, Hao P, Su J, Nan P, Feng Y, Shi YX, Yu XJ, Luo XM, Chen KX, Shen X, Shen JH, Zou JP, Zhao GP, Shi TL, He WZ, Zhong Y, Jiang HL, Li YX. 2003 Identification of probable genomic packaging signal sequence from SARS-CoV genome by bioinformatics analysis. Acta Pharmacol Sin 24 489-496.
29. Voss D, Kern A, Traggiai E, Eickmann M, Stadler K, Lanzavecchia A, Becker S. 2006. Characterization of severe acute respiratory syndrome coronavirus membrane protein. FEBS Lett 580:968-973.
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49518-
dc.description.abstract中東型呼吸道症候群 (Middle East Respiratory Syndrome)首發病例於2012年9月發現,之後經分析確定為新種冠狀病毒感染所引起之致命呼吸道疾病,並命名為中東型呼吸道症候群冠狀病毒 (Middle East Respiratory Syndrome Coronavirus,MERS-CoV)。冠狀病毒為帶有正股RNA基因體及套膜的病毒,其基因體約26-32 kb,可表現病毒複製時所需的非結構性蛋白質及結構性蛋白質,其中結構蛋白質N被認為是與其基因體結合的主要蛋白質。實驗室先前於SARS-CoV的研究中,發現SARS-CoV的RNA包裹訊號位於其基因體第19888-19950個核苷酸之間,且SARS-CoV可透過N蛋白質與此長63個核苷酸的RNA包裹訊號結合,進而被包裹至類病毒顆粒內。本篇論文的目的為探討MERS-CoV的RNA包裹訊號及MERS-CoV N蛋白質對於包裹RNA genome的重要性。將表現MERS-CoV四種結構蛋白質的質體轉染入Huh7細胞株中,轉染三天後收集其細胞及細胞培養液,以超高速離心的方式沉澱,建立類病毒顆粒的產出和純化系統。同時,透過基因體之序列及二級結構分析,推測MERS-CoV RNA包裹訊號位於基因體19712-19969的258個核苷酸片段。進一步收集帶有此258個核苷酸片段及EGFP序列RNA (EGFP-PS)的類病毒顆粒,感染帶有MERS-CoV receptor DPPIV的Huh7細胞株,證實此258個核苷酸片段確實帶有MERS-CoV RNA包裹訊號。當將表現EGFP-PS RNA 及MERS-CoV M、E及S蛋白質之質體同時轉染入Huh7細胞中時,發現不帶有N蛋白質的類病毒顆粒,在感染Huh7細胞株時無法偵測到EGFP蛋白質的表現,顯示MERS-CoV的RNA包裹需有N蛋白質的參與。未來可進一步探討具有RNA包裹功能的最小基因體RNA片段及重要的結構與序列,以及N蛋白質與RNA包裹訊號結合的區域。zh_TW
dc.description.abstractThe first case of Middle East respiratory syndrome was found in September 2012, Saudi Arabia. Laboratory testing identified this case as a novel coronavirus infection. The coronavirus is now later named Middle East respiratory syndrome coronavirus (MERS-CoV). Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with genomic RNA about 26-32 kb in length. The viral particle consists of four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). N protein is considered to be the most important protein that interacts with the viral genome. Our laboratory has previously demonstrated that SARS-CoV RNA packaging signal is located in the RNA fragment of SARS-CoV genome from nt 19888-19950. The RNA fragment can be assembled into virus-like particles (VLPs) in a N protein-dependent manner. The purpose of this study is to determine the RNA packaging signal of MERS-CoV and the involvement of N protein in the package of viral genome. To establish a system for producing MERS-VLPs, plasmids encoding the four structural proteins of MERS-CoV were co-transfected into Huh7 cells. Three days post-transfection, VLPs were collected and purified from the transfected cells and culture medium following ultracentrifugation. Meanwhile, the sequence and secondary structure of MERS-CoV genome were analyzed for prediction of MERS-CoV RNA packaging signal. VLPs carrying RNA sequence of EGFP fused to the putative MERS-CoV RNA packaging signal spanning nt 19712-19969 of the viral genome were used to incubate with MERS-CoV-permissive Huh7 cells. The results demonstrated that the 258-bp RNA sequence is sufficient to function as a packaging signal and be packaged into the MERS-VLPs. In addition, MERS-CoV RNA was packaged into the VLPs in a N protein-dependent manner. The RNA-interacting domain of the viral N protein and the minimal RNA sequences and structures critical for the viral genome package need to be future determined.en
dc.description.provenanceMade available in DSpace on 2021-06-15T11:32:36Z (GMT). No. of bitstreams: 1
ntu-105-R03445125-1.pdf: 2749078 bytes, checksum: af93c968ef947949cd75940cc13b6c75 (MD5)
Previous issue date: 2016
en
dc.description.tableofcontents摘要.................................................................i
Abstract..........................................................ii
目錄................................................................iv
壹、緒論...........................................................1
一、中東型呼吸道症候群冠狀病毒發現歷史 1
二、中東型呼吸道症候群冠狀病毒之感染症狀及流行病學 2
三、中東型呼吸道症候群冠狀病毒蛋白質 2
四、冠狀病毒之基因體結構及複製 4
五、冠狀病毒生活史 7
六、研究目的 7
貳、實驗材料與方法......................................9
一、 材料 9
二、 方法 16
參、實驗結果.................................................25
一、MERS-CoV RNA包裹訊號之預測 25
二、預測之MERS-CoV RNA包裹訊號二級結構分析 25
三、建構MERS-CoV RNA包裹訊號表現系統 26
四、MERS-CoV結構蛋白質於細胞中表現狀態 26
五、建立MERS-CoV類病毒顆粒系統 26
六、利用MERS-CoV類病毒顆粒系統進行RNA包裹訊號功能檢測 27
七、類病毒顆粒感染造成細胞形態改變 28
八、MERS-CoV RNA包裹具有冠狀病毒間的特異性 28
九、MERS-CoV RNA包裹需依靠N蛋白質與RNA包裹訊號結合 29
肆、討論.........................................................31
一、MERS-CoV結構蛋白質之修飾是否影響病毒顆粒形成及釋放 31
二、MERS-CoV類病毒顆粒感染造成細胞形態改變 31
三、MERS-CoV RNA包裹訊號之結構與序列之重要性 32
四、MERS-CoV N蛋白質對於RNA包裹之重要性 32
伍、圖表.........................................................34
陸、參考文獻................................................48
柒、附錄.......................................................52
dc.language.isozh-TW
dc.title中東型呼吸道症候群冠狀病毒RNA包裹訊號zh_TW
dc.titleRNA Packaging Signal of Middle East Respiratory Syndrome Coronavirusen
dc.typeThesis
dc.date.schoolyear104-2
dc.description.degree碩士
dc.contributor.oralexamcommittee詹世鵬(Shih-Peng Chan),董馨蓮(Shin-Lian Doong)
dc.subject.keyword中東行呼吸道症候群冠狀病毒,RNA包裹訊號,核殼蛋白質,zh_TW
dc.subject.keywordMERS-CoV,RNA packaging signal,nucleocapsid protein,en
dc.relation.page61
dc.identifier.doi10.6342/NTU201602880
dc.rights.note有償授權
dc.date.accepted2016-08-17
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept微生物學研究所zh_TW
顯示於系所單位:微生物學科所

文件中的檔案:
檔案 大小格式 
ntu-105-1.pdf
  目前未授權公開取用
2.68 MBAdobe PDF
顯示文件簡單紀錄


系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。

社群連結
聯絡資訊
10617臺北市大安區羅斯福路四段1號
No.1 Sec.4, Roosevelt Rd., Taipei, Taiwan, R.O.C. 106
Tel: (02)33662353
Email: ntuetds@ntu.edu.tw
意見箱
相關連結
館藏目錄
國內圖書館整合查詢 MetaCat
臺大學術典藏 NTU Scholars
臺大圖書館數位典藏館
本站聲明
© NTU Library All Rights Reserved