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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 齊肖琪 | |
dc.contributor.author | Pei-Yun Tsai | en |
dc.contributor.author | 蔡佩芸 | zh_TW |
dc.date.accessioned | 2021-06-16T17:18:34Z | - |
dc.date.available | 2014-08-27 | |
dc.date.copyright | 2012-08-27 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2012-08-17 | |
dc.identifier.citation | Akira, S., Uematsu, S., Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell, 124(4), 783-801.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63765 | - |
dc.description.abstract | 神經壞死病毒是魚類神經壞死病毒症的病原。罹患神經壞死病毒症之後的殘活魚,其中樞神經系統以及鰭部位皆可測到大量病毒核酸。為瞭解神經壞死症病毒與石斑魚先天性免疫防禦機制的交互關係,本實驗以石斑魚鰭細胞株GF-3為系統,研究干擾素誘導的抗病毒蛋白質Mx與神經壞死症病毒複製之間的關係。當GF-3細胞感染神經壞死病毒或轉染Poly I:C後,皆會誘發Mx蛋白質的表現, 並且Mx蛋白具有抗神經壞死病毒的活性。在神經壞死病毒感染的GF-3 細胞中,Mx蛋白會隨著感染時間而表現量會上升, 但病毒的RdRp (RNA-dependent RNA polymerase) 表現量則是下降。在感染神經壞死病毒 24小時的細胞中,用免疫螢光染色法可觀察到Mx 蛋白與在粒線體進行複製的NNV RdRp一起座落在細胞質中,同時間RdRp與lysosome的分佈位置也有重疊,因此推測,石斑魚細胞中的Mx蛋白可能藉由與RdRp結合並運送到lysosome進行降解以干擾病毒的複製。以低病毒感染劑量感染GF-3細胞,會形成病毒持續性感染的細胞株,每代細胞上清液中的病毒力價平均為105 TCID50 ml-1,但每代只有0.1% 的細胞有表現病毒蛋白,因此推測,GF-3細胞上清液存在類干擾素的細胞激素,且所誘導細胞表現的抗病毒Mx蛋白,可在GF-3細胞株的NNV持續性感染機制中扮演其中一個重要角色。 | zh_TW |
dc.description.abstract | Nervous Necrosis virus (NNV) is the causative agent of Viral Nervous Necrosis (VNN) disease among many species of fish. Groupers survived from VNN show high viral copies in the central nervous system and fin. However, the interplay between betanodavirus and the innate system defense of host remains unclear. The GF-3 cell line derived from grouper fin tissue was used to examine the interaction of interferon (IFN)-induced Mx protein and betanodavirus replication. The expression of grouper Mx Ⅱ was found after the infection with NNV or transfection with Poly I:C. IFN response and Mx protein showed antiviral activity in GF-3 cells. In addition, the expression of grouper Mx protein increased while viral RdRp (RNA-dependent RNA polymerase) decreased through the time courses of NNV life cycle. Grouper Mx was found in the cytoplasm at 24 h post infection (hpi) and co-localized with RdRp which replicates at mitochondria. Meanwhile, RdRp was found co-localized with lysosome. It is therefore suggested that Mx protein may interfere with NNV replication via binding with viral RdRp at mitochondria and translocate together to the lysosome for degradation. Besides, low multiplicity of NNV infection induced IFN response and Mx in GF-3 cells, and then the cells became NNV persistent infection (PI). Only 0.1% of the cells showed positive signal in the immunoflourescent staining with anti-capsid polyclonal antibodies, and the viral titer in the PI cell supernatant of each subculture was about 105 TCID50 ml-1. Therefore, the mechanism of NNV-PI in GF-3 cells is suggested to relate to IFN response and Mx protein. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:18:34Z (GMT). No. of bitstreams: 1 ntu-100-R99b41031-1.pdf: 2147307 bytes, checksum: 4f43b70e8026c16766fd1005bdebe7df (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要 I
Abstract II Contents IV Contents of Tables and Figures XI Introduction 1 1. Fish nodavirus 1 1.1 Clinical signs of viral nervous necrosis (VNN) 1 1.2 The characteristics of nervous necrosis virus (NNV) 2 1.3 Taxonomy and phylogeny of NNV 2 1.4 Host range and geographical distribution of VNN 3 2. Innate immune system 3 2.1 Pattern recognition receptors 3 2.2 Interferon system 5 2.3 Signaling pathways of IFN action 6 2.4 Interferon-stimulated genes 6 2.5 Mx proteins 7 3. The purpose of the study 9 Materials and Methods 11 1. Cell line and virus 11 2. Detection of NNV by RT- PCR and semi-nested PCR 11 3. Detection of grouper Mx mRNA by real-time PCR 13 4. Detection of grouper Mx protein by Western blot 14 5. Cloning of grouper Mx cDNA 14 5.1. RNA extraction, RT-PCR and semi-nested PCR 14 5.2. Ligation 15 6. Profiles of NNV titers in GF-1 and GF-3 cell lines during time coursesGF-1 cells collected and titrated in GF-1 cells. 17 7. Antiviral activity assay 17 8. Down-regulation of Mx gene expression by siRNA transfection 17 9. Detection of grouper Mx protein, NNV RdRp and capsid protein by Western blot 18 10. Immunofluorescence staining 19 11. Detection of NNV and Mx gene expression in NNV-persistently infected GF-3 cells ……………………………………………………………………………………..20 12. Comparison of cytopathic effect (CPE) in GF-3 cells and PI-GF-3 cells caused by NNV infection 21 Results 22 1. Detection of NNV RNA and Mx protein in NNV- infected GF-3 cells 22 2. Sequence analysis of the GF-3 Mx gene…………………………………………..22 3. The profile of viral titers in NNV infected GF-1 cells and GF-3 cells 23 4. Antiviral activity of IFN response in GF-3 cells 23 5. The impact of grouper Mx protein on the progeny virion 23 6. The expression of GF-3 Mx protein and viral proteins during time courses. 24 7. The localization of grouper Mx protein, NNV RdRp and capsid protein in NNV- infected GF-3 cells 24 8. The localization of viral RdRp, capsid, mitochondria, and lysosome 25 9. Low MOI induced NNV-persistent infection in GF-3 cells 25 10. NNV persistently infected (PI) GF-3 cells showed Mx gene expression 26 11. Comparison of cytopathic effect (CPE) in NNV-infected GF-3 cells and NNV re-infected PI-GF-3 cells 26 Discussion 27 References 32 Contents of Tables and Figures Table 1. Primers used in the experiment………………………………………………..43 Table 2. The comparison of GF-3 Mx with three isoforms of grouper Mx 44 Fig. 1. Detection of NNV RNA in NNV-infected GF-3 cells by RT-PCR.. 45 Fig. 2. Detection of Mx mRNA and Mx protein.. 46 Fig. 3. Variation of NNV titers in GF-1 and GF-3 cell lines at different time courses post infection.. 47 Fig. 4. Poly I:C-induced IFN response exhibited anti-NNV activity in GF-3 cells. 48 Fig. 5. Down-regulation of Mx expression by siRNA and the influence on viral replication…. .49 Fig. 6. The expression levels of GF-3 Mx, viral RdRp, and capsid proteins through the time courses of NNV infection (MOI=100) by Western blot. 51 Fig. 7. Detection of the distribution of Mx and viral proteins by immunofluorescence staining. 52 Fig. 8. The localization of NNV RdRp, mitochondria, and lysosome in NNV-infected or mock-infected GF-3 cells were detected by immunofluorescence staining. 53 Fig. 9. The morphology of GF-3 cells and NNV-infected GF-3 cells with MOI 0.1 and 1 at 7 dpi... 54 Fig. 10. Detection of NNV RNA2 by RT-PCR and nested-PCR in NNV-PI-GF-3 cells at 5th subculture...................................................................................................55 Fig. 11. Viral titers in the supernatant of NNV PI-GF-3 cells within 22 passages…….56 Fig. 12. Detection of NNV-positive cells in NNV PI-GF-3 cells by immunofluorescence staining.. 57 Fig. 13. The Mx gene expression level in GF-3 cells and NNV-persistently infected GF-3 cells.………………………………………………………………...…58 Fig. 14. The CPE of NNV-infected GF-3 and NNV-reinfected PI-GF-3 cells (MOI=100) at 7 days post infection………………………………………………………59 | |
dc.language.iso | zh-TW | |
dc.title | 石斑魚鰭細胞株GF-3 Mx蛋白質抗魚類野田病毒的機制 | zh_TW |
dc.title | The mechanism of grouper Mx protein against betanodavirus replication in GF-3 cell line | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張繼堯,張麗冠,陳歷歷 | |
dc.subject.keyword | 石斑魚,Mx蛋白質,魚類野田病毒, | zh_TW |
dc.subject.keyword | grouper,Mx protein,betanodavirus,NNV, | en |
dc.relation.page | 59 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-17 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 動物學研究所 | zh_TW |
顯示於系所單位: | 動物學研究所 |
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