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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 蕭信宏教授(Shin-Hong Shiao) | |
| dc.contributor.author | Yu-Xian Zhou | en |
| dc.contributor.author | 周育賢 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:32:55Z | - |
| dc.date.copyright | 2021-08-31 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-24 | |
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D., Henrion-Lacritick, A., Gausson, V., Suzuki, Y., Shengjuler, D., Meyer, B., Vallet, T., Weger-Lucarelli, J., Bernhauerova, V., Titievsky, A., Sharov, V., Pietropaoli, S., Diaz-Salinas, M. A., Legros, V., Pardigon, N., Barba-Spaeth, G., Brodsky, L., Saleh, M. C., and Vignuzzi, M. (2021) Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts. Nat Commun 12 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82153 | - |
| dc.description.abstract | "病媒蚊是許多重大疾病的傳播媒介，包含瘧疾、登革熱以及茲卡病毒感染症等等。其中登革熱作為世界上傳播速度最快的蚊媒傳染疾病之一，全世界約有一半的人口生活在登革熱流行的區域之中。隨著全球暖化，登革熱在世界各地蔓延的情形有愈發嚴峻的趨勢。由於登革熱目前尚無安全有效的疫苗可供接種，亦無藥物可供治療，因此，控制病媒蚊族群的數量為主要的防治策略，以減緩疾病的傳播。而噴灑殺蟲劑為目前病媒蚊防制的主要方法，然而，隨著具有抗藥性病媒蚊的增加，積極研發新興抗病毒策略便成了當務之急。在本研究中，我們在埃及斑蚊細胞株(ATC10)中建立一個能夠受黃病毒誘導之基因表達系統，並且利用此系統作為新興的抗病毒方式。首先我們以第二型登革病毒的五端及三端未轉譯蛋白區域序列之負股選殖至表現載體中，並加入登革病毒的RNA聚合酶基因 (Non-structural protein 5 RNA-dependent RNA-polymerase, NS5 RdRp)。並且以綠色螢光蛋白(Green fluorescent protein, GFP)基因作為報導基因 (Reporter gene)。我們的結果顯示，當帶有病毒誘導表達系統之細胞分別被四種血清型的登革病毒以及茲卡病毒感染後，細胞內GFP的表現量隨之顯著上升，顯示此系統可被不同黃病毒科的病毒所啟動。接著我們將此系統中的GFP置換成能夠誘導蚊子細胞凋亡的兩個基因，Michelob_x (AaMx)以及IAP-antagonist Michelob_x like protein (AaIMP)，並且測試此兩種系統的抗病毒能力。實驗結果顯示，當帶有病毒誘導AaMx或AaIMP表達系統之細胞分別被四種血清型的登革病毒以及茲卡病毒感染後，細胞內的細胞凋亡酶(Caspase-3)活性有顯著的上升。我們也發現此兩種細胞分別感染四種血清型的登革病毒以及茲卡病毒後，細胞內病毒RNA以及蛋白質的表現量相較於控制組有顯著的降低。除此之外，我們亦利用Focus-forming assay (FFA)偵測帶有病毒誘導系統之細胞在感染病毒後產生具有感染力的病毒的效價。實驗結果發現，三種帶有病毒誘導系統的細胞(GFP, AaMx, and AaIMP)在分別感染四種血清型的登革病毒以及茲卡病毒後，產生具有感染力的病毒的效價相較於控制組有顯著的降低。上述的實驗結果顯示了此病毒誘導系統的抗病毒能力，除此之外，我們也初步確認帶有病毒誘導系統的蚊子對於第二型登革病毒具有顯著的抑制效果。我們的研究顯示病毒誘導系統可以成功的抑制登革病毒及茲卡病毒的複製及傳播，可提供未來建構新型抗病毒策略的參考模式。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:32:55Z (GMT). No. of bitstreams: 1 U0001-2308202114302500.pdf: 3503955 bytes, checksum: 6b246a7eb4411e4f445111ae20a326a7 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | Acknowledgement i 中文摘要 iii Abstract v Table of Contents vii List of Figures xi List of Tables xiii Chapter 1. Introduction 1 1.1. Mosquito-borne disease 1 1.2. Mosquito control strategies 3 1.3. Dengue 5 1.4. Flavivirus infectious life cycle 6 1.5. Apoptosis 8 1.6. Bacterial delivery approach 9 1.7. Specific aims and hypothesis 10 Chapter 2. Materials and Methods 11 2.1. Plasmid construction 11 2.1.1. Generation of the antisense mini-replicon of GFP 11 2.1.2. Generation of the pUC-NS5-GFP 13 2.1.1. Generation of the pUC-NS5-IMP and pUC-NS5-Mx 13 2.2. Cell culture 14 2.3. Stable transfection 15 2.4. Mosquito 15 2.5. Bacterial delivery system 16 2.6. Virus and infection 17 2.7. Genomic DNA (gDNA) isolation 18 2.8. RNA isolation 19 2.9. Reverse transcription (RT) 19 2.10. Diagnostic polymerase chain reaction (Diagnostic PCR) 20 2.11. Quantitative polymerase chain reaction (qPCR) 20 2.12. Immunofluorescence assay (IFA) 21 2.13. Caspase-3 activity assay 22 2.14. Focus-forming assay (FFA) 23 2.15. Crystal violet assay 24 Chapter 3. Results 26 3.1. Establishment of the virus-inducible gene expression system 26 3.2. The flavivirus-inducible gene expression system is activated after different flaviviruses infection. 27 3.3. The flavivirus-inducible gene expression system inhibited the viral genome replication after different flaviviruses infection. 29 3.4. The flavivirus-inducible gene expression system inhibited the expression of viral NS1 protein after different flaviviruses infection. 30 3.5. The flavivirus-inducible gene expression system limited the production of infectious virus particles after different flaviviruses infection. 31 3.6. The antiviral activity of transfected ATC10 cell lines against flaviviruses is not due to the growth defects in virus infected cells. 32 3.7. The flavivirus-inducible gene expression system promotes antiviral activity in mosquitoes. 33 3.8. The flavivirus-inducible gene expression system was not observed in the offspring of mosquitoes carrying virus-inducible system. 34 Chapter 4. Discussion 36 Figures 42 Tables 64 References 67 | |
| dc.language.iso | en | |
| dc.subject | 埃及斑蚊 | zh_TW |
| dc.subject | 登革病毒 | zh_TW |
| dc.subject | 茲卡病毒 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 病毒誘導基因表現系統 | zh_TW |
| dc.subject | dengue virus | en |
| dc.subject | virus-inducible gene expression system | en |
| dc.subject | Zika virus | en |
| dc.subject | Aedes aegypti | en |
| dc.subject | apoptosis | en |
| dc.title | 建構黃病毒誘導基因表達系統作為新興抗病毒策略 | zh_TW |
| dc.title | Establishment of a flavivirus-inducible gene expression system as a novel antiviral approach | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 洪健清主任(Chien-Ching Hung) | |
| dc.contributor.oralexamcommittee | 顧家綺副教授(Hsin-Tsai Liu),楊宏志副教授(Chih-Yang Tseng) | |
| dc.subject.keyword | 埃及斑蚊,登革病毒,茲卡病毒,細胞凋亡,病毒誘導基因表現系統, | zh_TW |
| dc.subject.keyword | Aedes aegypti,dengue virus,Zika virus,apoptosis,virus-inducible gene expression system, | en |
| dc.relation.page | 87 | |
| dc.identifier.doi | 10.6342/NTU202102620 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-08-24 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-08-23 | - |
| Appears in Collections: | 微生物學科所 | |
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| U0001-2308202114302500.pdf Restricted Access | 3.42 MB | Adobe PDF | View/Open |
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