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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳育騏 | zh_TW |
| dc.contributor.advisor | Yu-Chi Wu | en |
| dc.contributor.author | 許渟 | zh_TW |
| dc.contributor.author | Ting Hsu | en |
| dc.date.accessioned | 2025-08-21T16:28:26Z | - |
| dc.date.available | 2025-08-22 | - |
| dc.date.copyright | 2025-08-21 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-05 | - |
| dc.identifier.citation | ․ Adachi, K., Ichinose, T., Takizawa, N., Watanabe, K., Kitazato, K., & Kobayashi, N. (2007). Inhibition of betanodavirus infection by inhibitors of endosomal acidification. Archives of virology, 152, 2217-2224.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99122 | - |
| dc.description.abstract | 雙股RNA激活蛋白激酶 (Double-stranded RNA-activated protein kinase, PKR) 為干擾素誘導基因之一，在脊椎動物先天免疫中扮演重要角色。PKR的典型抗病毒機制，為透過磷酸化eIF2α、抑制蛋白質轉譯，在抑制病毒蛋白表現的同時，也引發細胞自噬避免病毒複製。此反應途徑已在哺乳動物及部分真骨魚類中證實；但在石斑魚中，PKR在病毒感染下的功能仍所知有限。本次自點帶石斑魚 (Epinephelus coioides) 細胞中分離出PKR同源物，並命名為GPKR；實驗旨在探討其於神經壞死病毒 (Nervous Necrosis Virus, NNV) 感染下的表現模式與功能，並進一步驗證其是否會影響細胞自噬。GPKR基因包含長度為1659 bp的ORF，對應552個胺基酸，在系統發育樹分析中，與龍膽石斑魚PKR最相近。GPKR蛋白具有N端的兩個dsRBM (Double-stranded RNA binding motif) 及C端的Serine/Threonine激酶結構域；因與其他脊椎動物PKR具有相似的結構域，因此推測GPKR可能也具有相似的功能。為探討GPKR在病毒感染下於活體中的表現，首先以107 TCID50之NNV攻毒石斑魚，並在感染前後以qPCR測定不同組織的GPKR mRNA表現量。結果顯示，GPKR廣泛存在於所有觀察的組織中，並在感染前就有基本表現量，感染後則在心臟、肝臟與脾臟中顯著上調。在細胞實驗中，以NNV感染石斑魚腦細胞 (cGB) 與鰭細胞 (GF-3)，觀察到GPKR的顯著上調，證實其會受病毒感染引發。此外若在細胞中轉染poly (I:C)，同樣能刺激GPKR的表現；進一步顯示其受dsRNA誘發的特性。若以siRNA敲落GPKR基因，則病毒NNV RNA2的表現量會在感染後72小時下降，顯示GPKR可能參與NNV的複製過程。為探討GPKR是否參與自噬反應，本次利用MDC染劑標記自噬小體，並觀察其數量變化。結果顯示自噬小體數量會隨病毒感染增多，但GPKR敲落並不會對自噬反應造成影響。本研究在點帶石斑魚中，首次分離並定義PKR同源蛋白：GPKR。有別於脊椎動物PKR常見的抗病毒功能，GPKR似乎參與了NNV的複製，且其作用機制與自噬反應無關。 | zh_TW |
| dc.description.abstract | Double-stranded RNA-activated protein kinase (PKR) is an interferon-stimulated gene that plays a crucial role in the innate immune response of vertebrates. Its classical antiviral mechanism involves phosphorylation of eIF2α, which inhibits protein translation, thereby suppressing viral protein synthesis and triggering autophagy to prevent viral replication. While this pathway has been well-documented in mammals and certain teleost species, the functional role of PKR during viral infection in groupers remains largely unknown. In this study, we identified and cloned a PKR homolog: GPKR from orange-spotted grouper (Epinephelus coioides) and investigated its expression pattern and potential function during nervous necrosis virus (NNV) infection. Its possible involvement in autophagy was also examined. GPKR gene contains an ORF of 1659 bp, encoding a protein of 552 amino acids. Phylogenetic analysis showed that GPKR is most closely related to giant grouper PKR. GPKR contained two N-terminal double-stranded RNA-binding motifs (dsRBMs) and a C-terminal serine/threonine kinase domain. This result indicated functional domain conservation and suggesting potential functional similarity between GPKR and other vertebrate PKRs. To evaluate the in vivo expression of GPKR, groupers were challenged with NNV at a dose of 10⁷ TCID₅₀. Tissue-specific GPKR mRNA levels were measured using qPCR before and after infection. Results showed that GPKR was found to be constitutively expressed in all examined tissues pre-infection and significantly upregulated in heart, liver, and spleen post-infection. In vitro, NNV infection of grouper brain cells (cGB) and fin cells (GF-3) resulted in upregulation of GPKR, confirming its inducibility by viral infection. Similarly, transfection with poly(I:C) also elevated GPKR levels, suggesting that its upregulation is responsive to double-stranded RNA stimulation. Knockdown of GPKR via siRNA led to a reduction in NNV RNA2 levels at 72 hours post-infection, indicating a potential role in supporting viral replication. To assess whether GPKR is involved in autophagy, MDC staining was used to monitor autophagosome formation. The results showed an increase in autophagosome number following viral infection; however, no significant difference of autophagosome number was found in GPKR knockdown cells. This is the first study to characterize a new PKR homolog: GPKR in E. coioides. Unlike the typical antiviral function of vertebrate PKRs, GPKR appears to play a role in NNV replication, possibly through mechanisms that are independent of autophagy. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-21T16:28:26Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-21T16:28:26Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 目次
致謝 I 中文摘要 II 英文摘要 IV 目次 VI 圖次 VIII 表次 VIII 第一章、文獻回顧 1 1. 神經壞死病毒 1 1.1 NNV基因組及基因型 1 1.2 病毒性神經壞死症 2 1.3 NNV感染後在組織中的分布 2 1.4 NNV的感染與複製 3 1.5 NNV之細胞病變效應 4 1.6 NNV感染引發的先天免疫反應 4 2. 雙股RNA激活蛋白激酶 5 2.1 PKR的結構與活化 5 2.2 PKR之抗病毒功能 6 2.3魚類的PKR 6 2.4 病毒的抗PKR機制 7 3. 細胞自噬 8 3.1細胞自噬簡介 8 3.2 細胞自噬與NNV 9 4. 點帶石斑魚 10 5. 研究動機 10 第二章、材料與方法 12 2.1 細胞株與病毒 12 2.2 GPKR序列分析 12 2.3 攻毒與病毒感染 13 2.4 RNA干擾 14 2.5 RNA萃取 14 2.6 反轉錄聚合酶連鎖反應 15 2.7 即時定量PCR 15 2.8 MDC染色和自噬小體偵測 15 2.9 統計方法 16 第三章、結果 17 3.1 GPKR之序列與系統發育樹分析 17 3.2 GPKR之保守結構域分析 18 3.3 GPKR之表現模式檢測 18 3.4 GPKR敲落對NNV複製的影響 20 3.5 GPKR敲落對細胞自噬的影響 20 第四章、討論 22 4.1 GPKR之相似度與系統發育樹分析 22 4.2 GPKR之多序列比對分析 23 4.3 GPKR與NNV在各組織間的表現模式 24 4.4 GPKR與NNV在細胞中的表現模式 26 4.5 PKR敲落效果的延遲 27 4.6自噬小體數量偵測：影像分析與螢光偵測法之比較 28 4.7 GPKR表現對NNV複製的影響 29 4.8 總結 30 第五章、參考文獻 32 表次 表1 本次研究中使用的引子 (primer) 與siRNA序列 42 表2 與GPKR全長序列比對的13種魚類與哺乳動物PKR之NCBI登錄號及相似度對照表 43 圖次 圖1. GPKR與其他13種魚類及哺乳動物PKR之系統發育樹 44 圖2. GPKR與其他7種魚類及哺乳動物PKR之保守結構域與多序列比對分析 45 圖3. 石斑魚感染前後各臟器中的GPKR與NNV RNA2表現量變化 50 圖4. NNV感染或poly (I:C) 轉染細胞中GPKR與NNV RNA2的表現量變化 52 圖5. GPKR敲落在0-72 hpi對NNV複製的影響 53 圖6. 以影像分析法與螢光偵測法檢測NNV感染下的細胞自噬反應 54 圖7. 以螢光測法觀察GPKR敲落在24 hpi對細胞自噬的影響 55 圖8. GPKR與其他13種魚類及哺乳動物PKR之 dsRBM系統發育樹分析 56 附圖1. NNV RNA2 Cq值換算複製量之對照圖表 57 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 雙股RNA激活蛋白激酶 | zh_TW |
| dc.subject | 神經壞死病毒 | zh_TW |
| dc.subject | 細胞自噬作用 | zh_TW |
| dc.subject | 點帶石斑魚 | zh_TW |
| dc.subject | 先天免疫 | zh_TW |
| dc.subject | autophagy | en |
| dc.subject | PKR | en |
| dc.subject | NNV | en |
| dc.subject | innate immunity | en |
| dc.subject | orange-spotted grouper | en |
| dc.title | 石斑魚雙股RNA激活蛋白激酶對野田病毒複製與細胞自噬之影響 | zh_TW |
| dc.title | The effect of grouper double-stranded RNA-activated protein kinase on Betanodavirus replication and autophagy | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 韓玉山;蓋玉軒 | zh_TW |
| dc.contributor.oralexamcommittee | Yu-Shan Han;Yu-Hsuan Kai | en |
| dc.subject.keyword | 雙股RNA激活蛋白激酶,神經壞死病毒,細胞自噬作用,點帶石斑魚,先天免疫, | zh_TW |
| dc.subject.keyword | PKR,NNV,autophagy,orange-spotted grouper,innate immunity, | en |
| dc.relation.page | 57 | - |
| dc.identifier.doi | 10.6342/NTU202503137 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-07 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 漁業科學研究所 | - |
| dc.date.embargo-lift | 2025-08-22 | - |
| 顯示於系所單位： | 漁業科學研究所 | |
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