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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78830完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張鑫 | zh_TW |
| dc.contributor.advisor | Shin C.Chang | en |
| dc.contributor.author | 張池佑 | zh_TW |
| dc.contributor.author | Chi-You Chang | en |
| dc.date.accessioned | 2021-07-11T15:22:29Z | - |
| dc.date.available | 2023-11-24 | - |
| dc.date.copyright | 2019-03-11 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78830 | - |
| dc.description.abstract | 中東呼吸症候群冠狀病毒(MERS-CoV)是於2012年出現的新型冠狀病毒。此病毒會造成嚴重的肺炎以及多重器官衰竭，總體感染死亡率約為35%。為了瞭解MERS-CoV的發病機制，有不少研究是在探討宿主的免疫系統與病毒致病力彼此之間的對抗關係。干擾素是一類重要的先天性免疫系統，它們能夠誘發上千種抗病毒基因的表現。在此篇研究中，首先分析MERS-CoV各結構蛋白質對干擾素表現的影響。藉由qRT-PCR的分析，發現膜蛋白質(M)與核殼蛋白質(N)都會抑制干擾素的表現。本研究針對N蛋白質進一步分析，證實此蛋白質能夠以劑量性地抑制β干擾素與λ干擾素的mRNA表現和它們的啟動子活性。GFP-辛德畢斯重組病毒增殖實驗驗證了N蛋白質抑制干擾素表現的作用。此外，過去已知RIG-I是一種辨識病毒RNA的蛋白質受器，經RIG-I的CARD被E3連接酶TRIM25泛素化，而促進干擾素的表現。本研究發現由RIG-I CARD誘導的干擾素啟動子活性與RIG-I/CARD的泛素化皆會因N蛋白質的存在而下降，這說明RIG-I的訊息傳遞途徑正是N蛋白質的干預目標。實驗中也確認了N蛋白質與TRIM25之間有交互作用、單獨的C端結構域N(237-413)就能抑制β干擾素啟動子的活性、TRIM25的過量表現復原了N蛋白質抑制干擾素的問題。N蛋白質只能影響RIG-I的訊息傳遞，而不會影響干擾素的訊息傳遞。我們研究出N蛋白質抑制干擾素的機制，讓我們更加瞭解了一個MERS-CoV的感染現象。 | zh_TW |
| dc.description.abstract | Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus having occurred since 2012. It causes severe pneumonia and multi-organ failure. The overall mortality rate of the virus infection is about 35%. To investigate the virus pathogenesis, researchers have focused on the interplay between the host immune defense and virulence. Interferon (IFN) is one of the important innate immune system triggering thousands of downstream antiviral genes. In this study, MERS-CoV structural proteins were examined for their effects on interferon expression. Both membrane protein (M protein) and nucleocapsid protein (N protein) were found to suppress IFN production in qRT-PCR analysis. The mechanism of N protein involved in interferon suppression was further examined. N protein dose-dependently suppressed IFN-β and IFN-λ mRNA expression and promoter activity. The down-regulation of interferon protein expression was functionally identified by implementing the rescue of recombinant Sindbis virus-GFP replication. RIG-I is a cytoplasmic RNA sensor recognizing viral RNA to induce interferon expression through being ubiquitinated on its CARDs by an E3 ligase, tripartite motif-containing protein 25 (TRIM25). The decline of RIG-I-CARD-induced IFN promoter activity and RIG-I/CARD ubiquitination in the presence of N protein also revealed that RIG-I pathway is the target which N protein disrupted. The N protein interacted with TRIM25. In addition, the C-terminal domain from amino acid 237 to 413, designated as N(237-413), suppressed IFN-β promoter activity. TRIM25 over-expression rescued the N protein-suppressed interferon promoter activity. Furthermore, MERS-CoV N protein had no effect on interferon-induced downstream gene expression. These results indicated that the N protein only blocked RIG-I pathway, but not interferon pathway. The study elucidates interferon suppression mechanism of MERS-CoV N protein leading to a further understanding on MERS-CoV pathogenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:22:29Z (GMT). No. of bitstreams: 1 ntu-108-R02445124-1.pdf: 2822083 bytes, checksum: 80311d4b72e7e636e538494e1e22d837 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 中文摘要 (i)
Abstract (ii) Content (iv) Content of figures (vii) Introduction (1) I. MERS-CoV (1) 1. History and virus features (1) 2. Origin and transmission (2) 3. Current treatment (3) 4. Virology (4) II. Innate immune response to RNA virus infection (8) III. RIG-I pathway (10) 1. RIG-I (10) 2. Ubiquitination and E3 ligase TRIM25 (10) IV. Interferon (11) Specific aim (14) Materials and Methods (16) I. Materials (16) 1. Chemicals and reagents (16) 2. Enzymes (18) 3. Antibodies (18) 4. Kits (19) 5. Cell lines (19) 6. Plasmids (19) II. Methods (23) 1. Agarose gel electrophoresis (23) 2. Transformation (23) 3. DNA transfection (24) 4. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (24) 5. Western blot analysis (25) 6. Production of lentivirus particles (26) 7. RNA extraction (26) 8. Quantitative real-time polymerase chain reaction (qRT-PCR) (26) 9. Luciferase assay (27) 10. Immunoprecipitation (IP), co-immunnoprecipitation (Co-IP), and ubiquitination assay (27) 11. Immunofluorescence assay (27) 12. Sendai virus and interferon induction (28) 13. Rescue of Sindbis virus-GFP (28) 14. Exogenous IFN-β treatment (28) Results (30) I. The effects of MERS-CoV structural proteins on IFN-α, IFN-β, and IFN-λ mRNA expression (30) II. MERS-CoV N protein rescues the replication of GFP recombinant Sindbis virus (30) III. MERS-CoV N protein suppresses IFN-β and IFN-λ promoter activity (31) IV. MERS-CoV N protein suppresses NF-κB activity (32) V. MERS-CoV N protein suppresses CARD-induced IFN-β and IFN-λ promoter activity (33) VI. MERS-CoV N protein reduces RIG-I and RIG-I CARD ubiquitination (34) VII. MERS-CoV N protein suppresses TRIM25-induced IFN-β and IFN-λ promoter activity (34) VIII. The interaction between MERS-CoV N protein and TRIM25 (34) IX. TRIM25 over-expression rescues MERS-CoV N protein-suppressed IFN-β 36 and IFN-λ promoter activity (35) X. The interaction between the linker region (165-236 aa)/C-terminal domain (237-413 aa) of MERS-CoV N protein and TRIM25 (36) XI. The C-terminal domain of MERS-CoV N protein suppresses IFN-β promoter activity (36) XII. The effects of MERS-CoV N protein on ISRE and CXCL10 promoter activity (37) XIII. Conclusion (38) Discussion (39) Figures (44) Reference (64) Appendix Table and Figures (78) | - |
| dc.language.iso | en | - |
| dc.subject | 核殼蛋白質 | zh_TW |
| dc.subject | TRIM25 | zh_TW |
| dc.subject | RIG-I | zh_TW |
| dc.subject | 泛素化 | zh_TW |
| dc.subject | 干擾素 | zh_TW |
| dc.subject | 核殼蛋白質 | zh_TW |
| dc.subject | 中東呼吸症候群冠狀病毒 | zh_TW |
| dc.subject | TRIM25 | zh_TW |
| dc.subject | RIG-I | zh_TW |
| dc.subject | 泛素化 | zh_TW |
| dc.subject | 干擾素 | zh_TW |
| dc.subject | 中東呼吸症候群冠狀病毒 | zh_TW |
| dc.subject | nucleocapsid protein | en |
| dc.subject | MERS-CoV | en |
| dc.subject | interferon | en |
| dc.subject | ubiquitination | en |
| dc.subject | RIG-I | en |
| dc.subject | TRIM25 | en |
| dc.subject | MERS-CoV | en |
| dc.subject | nucleocapsid protein | en |
| dc.subject | interferon | en |
| dc.subject | ubiquitination | en |
| dc.subject | RIG-I | en |
| dc.subject | TRIM25 | en |
| dc.title | 中東呼吸症候群冠狀病毒核殼蛋白質與TRIM25交互作用抑制干擾素β與λ的表現 | zh_TW |
| dc.title | MERS-CoV nucleocapsid protein suppresses IFN-β and IFN-λ expression by interacting with TRIM25 | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 鄧述諄;伍安怡 | zh_TW |
| dc.contributor.oralexamcommittee | Shu-Chun Teng;An-Ye Wu | en |
| dc.subject.keyword | 中東呼吸症候群冠狀病毒,核殼蛋白質,干擾素,泛素化,RIG-I,TRIM25, | zh_TW |
| dc.subject.keyword | MERS-CoV,nucleocapsid protein,interferon,ubiquitination,RIG-I,TRIM25, | en |
| dc.relation.page | 91 | - |
| dc.identifier.doi | 10.6342/NTU201900429 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-02-12 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| 顯示於系所單位： | 微生物學科所 | |
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