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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳媺玫 | |
dc.contributor.author | Ting-Hsuan Hsu | en |
dc.contributor.author | 許庭瑄 | zh_TW |
dc.date.accessioned | 2021-06-17T02:16:03Z | - |
dc.date.available | 2023-01-04 | |
dc.date.copyright | 2018-01-04 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-10-12 | |
dc.identifier.citation | Adamek, M., Rakus, K.L., Brogden, G., Matras, M., Chyb, J., Hirono, I., Kondo, H., Aoki, T., Irnazarow, I., Steinhagen, D., 2014a. Interaction between type I interferon and Cyprinid herpesvirus 3 in two genetic lines of common carp Cyprinus carpio. Dis Aquat Organ 111, 107-118.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68263 | - |
dc.description.abstract | 錦鯉疱疹病毒 (cyprinid herpesvirus 3, CyHV-3, KHV) 為Cyprinivirus屬,Alloherpesviridae科,疱疹病毒目( Herpesvirales)的成員。其為一帶有封套的雙股 DNA病毒,病毒大小約為 167-200 nm,核酸序列長度為 295 kbp (Michel et al., 2010a; Rakus et al., 2013)。 CyHV-3對鯉魚(Cyprinus carpio )、錦鯉(Cyprinus carpio haematopterus )以及鯉魚之雜交品種具有高傳染性、高致死性(Ouyang et al., 2013),對鯉魚養殖業造成重大的危害(Ouyang et al., 2013)。
如同其他疱疹病毒,CyHV-3也會有潛伏感染,而潛伏感染通常與免疫逃脫有關,Aoki等人曾在2007年對CyHV-3進行全基因分析,發現了五個與免疫逃脫有關的蛋白,分別為G-protein coupled receptor (encoded by ORF16),TNF receptor homologues (ORF4 and ORF12),E3L like protein(encoded by ORF112) 及 cyhv3IL-10 (ORF134) (Aoki et al., 2007)。Ouyang等人於2013年透過蛋白質體分析,發現感染CyHV-3魚隻體內的病毒蛋白以ORF12與ORF134蛋白表現量最大(Ouyang et al., 2013),加上已有多篇論文證實IL-10與病毒潛伏有關(Ouyang et al., 2014)。因此本研究的目的為評估cyhv3IL-10在魚體感染疱疹病毒後,是否參與發炎反應的調控以及與病毒潛伏的關係。 本實驗以重組蛋白cyhv3IL-10( rvIL-10) 分別進行體內(In vivo)以及體外( In vitro) 的實驗。在in vitro實驗中,以錦鯉巨噬細胞所產生的IL-12基因以及IL-10下游基因socs-3來評估rvIL-10蛋白的活性。在in vivo實驗中以naïve 及carrier魚感染錦鯉疱疹病毒72hr ,給予rvIL-10蛋白,每天觀察魚隻及其臨床症狀,並利用即時定量聚合酶鏈鎖反應(Real-Time PCR)檢測鰓、腎臟、脾臟的病毒量以及脾臟的免疫相關細胞激素的基因表現。 研究發現,給予具有活性的rvIL-10蛋白後,魚隻的臨床症狀較不明顯,病毒的複製也受到限制。在免疫的調節上,Th1免疫反應以及發炎相關免疫調控皆有延後的現象,推測是因 rvIL-10重組蛋白具有免疫抑制功能,導致錦鯉免疫反應延後,病毒複製受到的抑制與免疫功能的抑制可能使得病毒進入潛伏感染,而讓施打rvIL-10的錦鯉症狀減緩。魚隻感染CyHV-3後給予rvIL-10可有效緩解錦鯉疱疹病毒所造成的症狀,這是本研究新的發現,然而,rvIL-10造成此現象的詳細機制仍需更進一步研究。 | zh_TW |
dc.description.abstract | The cyprinid herpesvirus 3 (CyHV-3, KHV) is taxonomically grouped within the genus of cyprius, the family Alloherpesviridae and the order of herpesvirales. The genome size of CyHV-3 is 295 kb and mature viral particles are 167 to 200 nm in diameter and are surrounded by double strand DNA envelope. CyHV-3 is highly contagious and highly lethal to Cyprinus carpio (Cyprinus carpio), koi (Cyprinus carpio haematopterus) and the common carp hybrids that cause serious impact to aquaculture.
CyHV-3, as the same as other herpesvirus, may cause inapparent infection which is associated with immune evasion. There are five proteins related to immune evasion discovery through genome analysis of CyHV-3 in 2007 by Aoki., et al. These proteins are G-protein coupled receptor (encoded by ORF16), TNFR homologues (encoded by ORF4 and ORF12), E3L like protein (encoded by ORF112) and an interleukine-10 (IL-10) homologue (encoded by ORF134). Furthermore, IL-10 is one of the most important anti-inflammatory cytokines with a key role in the termination of inflammation and restoration of homeostasis, suggesting that CyHV-3 IL-10 might have functions on host immunosuppression, resulting that virus can’t be completely removed by host. The important immunomodulatory functions that ensure viral persistence in the host, then became carriers, cause persistent infection. Persistent infection is a major risk for CyHV-3 prevention, thus causing the virus to spread quickly, resulting in a serious lethality. Therefore, this is mainly to study cyhv3IL-10 protein. In this study, cyhv3IL-10 recombinant protein (rvIL-10) is applied in the in vivo experiment to verify the function of rvIL-10 in koi inflammatory response and anti-viral pathways immune mechanism regulation. The in vitro study, IL-12 gene and IL-10 accessory gene socs-3 produced by macrophage are measured to evaluate the bioactivity of cyhv3IL-10 proteins. The results showed that after the administration of rvIL-10 protein, the death number of koi was less than the control group, and the virus replication is also limited. In addition, the regulations of cell-mediated immune response present a delayed phase. In conclusion, this study provided a new insight that rvIL-10 may have the potential to be used to reduce the symptoms produced after CyHV-3 infection. However, the mechanism of rvIL-10 regulations still needs further studies. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:16:03Z (GMT). No. of bitstreams: 1 ntu-106-R04629021-1.pdf: 3718684 bytes, checksum: 5f286bab5b6369822e1e2492d9ee9c25 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 摘要 I
Abstract II 目錄 IV 圖目錄 1 表目錄 2 第一章 緒論 3 第二章 文獻回顧 5 第一節 錦鯉疱疹病毒之簡介 5 1.1 病毒型態與命名分類 5 1.2 病毒之分子結構與複製 6 1.3 宿主與發病因子 6 1.4 臨床症狀、剖檢病變與病理變化 7 1.5 傳染途徑與傳播方式 7 1.6 診斷方式 8 1.7 錦鯉疱疹病毒疫苗的發展狀況 8 第二節 魚類的免疫學 9 2.1 魚類免疫學 9 2.2 魚類對病毒感染的免疫反應 10 第三節 疱疹病毒的感染 12 3.1 疱疹病毒感染所引起的免疫反應 12 3.2 錦鯉疱疹病毒感染的免疫調控機制 14 第四節 介白素10(Interlukin10) 17 4.1 介白素介紹 17 4.2 鯉魚的IL-10 (Interleukin 10) 20 4.3 疱疹病毒的介白素10(vIL-10) 21 第三章 材料與方法 24 第一節 實驗設計與流程 24 第二節 CyHV-3感受性細胞培養與定量分析 25 2.1 Naïve 魚隻的檢測 25 2.2 細胞培養與繼代 25 第三節 病毒的培養與定量 27 3.1 病毒增殖 27 3.2 病毒的定量 27 3.3 CyHV-3 病毒液進行聚合酶鏈鎖反應(PCR)檢測 28 第四節 rvIL-10蛋白質的純化與生物活性檢測 32 4.1 錦鯉巨噬細胞的培養 32 4.2 蛋白質的純化與確認 33 4.3 rvIL-10的定量與定性分析 34 4.4 RNA萃取 35 4.5 cDNA的合成(Reverse transcription) 36 4.6 Real Time-PCR(Real-time polymerase chain reaction,即時聚合酶鏈鎖反應) 37 第五節 rvIL-10蛋白對CyHV-3感染魚的影響 39 5.1 攻毒前的檢測 39 5.2 魚隻攻毒與腹腔注射蛋白(rvIL10蛋白) 39 5.3 攻毒後的魚隻進行組織的RNA萃取以及cDNA的合成 41 5.4 利用Real-time PCR進行免疫反應檢測 41 5.5 死亡魚隻的檢測 43 第四章 結果 44 第一節 建立對CyHV-3具有感受性的細胞株 44 1.1 Naïve 與carriers的檢測 44 1.2 建立初代細胞以及繼代 44 1.3 初代細胞感受性檢測: 45 1.4 CyHV-3病毒定量 45 第二節 rvIL-10蛋白質的製備 46 第三節 利用體外培養巨噬細胞進行rvIL-10蛋白的活性檢測(in vitro) 46 3.1 錦鯉巨噬細胞初代培養的建立 46 3.2 rvIL-10的活性檢測 46 第四節 rvIL-10對CyHV-3感染魚七天內的影響 47 4.1 naïve 魚隻攻毒CyHV-3的臨床症狀 47 4.2 病毒DNA遺傳物質定量檢測 48 4.3 naïve 魚之免疫相關基因表現量檢測 48 第五節 rvIL-10對感染CyHV-3魚隻後期的影響 49 5.1 naïve 魚隻攻毒CyHV-3的臨床症狀 49 5.2 病毒DNA遺傳物質定量檢測 49 5.3 naïve 魚之免疫相關基因表現量檢測 50 第六節 帶原魚攻毒CyHV-3病毒後,給予rvIL-10後的差異 51 6.1 帶原魚隻攻毒CyHV-3與臨床症狀 51 6.2 病毒DNA遺傳物質定量檢測 52 6.3 帶原魚之免疫相關基因表現量檢測 52 第五章 討論 53 第一節 rvIL-10蛋白在巨噬細胞上的生物活性檢測 53 第二節 錦鯉疱疹病毒vIL-10在naïve 魚體內的免疫分析 54 第三節 帶原魚隻攻毒後給予rvIL-10隻免疫分析 59 第六章 結論 62 表格 1. 帶有vIL-10的病毒 63 圖 1 外來抗原所引起的宿主免疫調控反應 64 圖 2 IL-10的免疫影響與調控 65 圖 3 TKF4初代細胞株的型態與病毒攻毒後的結果 66 圖 4 錦鯉疱疹病毒的診斷 67 圖 5 以SDS PAGE與Western blot來確認rvIL-10表現 68 圖 6 錦鯉巨噬細胞的培養與檢測 69 圖 7 rvIL-10的生物活性檢測 70 圖 8 Naïve 魚隻感染錦鯉疱疹病毒七天內的檢測實驗 71 圖 9 Naïve 魚隻症狀差異圖 72 圖 10 Naïve 魚組織病理切片(H&E染色) 73 圖 11 Naïve 魚隻病毒攻毒後第三天至第七天內病毒copy number定量 74 圖 12 vIL-10基因表現量分析 75 圖 13 Naïve 魚隻免疫相關細胞激素基因表現量分析 77 圖 14 Naïve魚隻進行錦鯉疱疹病毒的慢性感染實驗 79 圖 15 Naïve 魚隻慢性感染的病毒copy number 定量 80 圖 16 Naïve 魚隻免疫相關細胞激素的基因檢測 81 圖 17 帶原魚病毒攻毒實驗 82 圖 18 帶原魚的臨床症狀分析 83 圖 19 帶原魚組織病理切片(H&E染色) 84 圖 20 帶原魚攻毒後第三天至第七天的病毒copy number定量 85 圖 21 帶原魚免疫相關細胞激素基因表現量分析 86 圖 22 帶原魚隻的PBS組別不同臟器在病毒量上的差異圖 87 圖 23 人類巨細胞病毒感染時期,病毒IL-10的免疫調控 88 參考文獻 89 | |
dc.language.iso | zh-TW | |
dc.title | 錦鯉疱疹病毒IL-10對細胞免疫調節激素的影響 | zh_TW |
dc.title | Effect of Recombinant Cyhv3IL-10 on Cell-Mediated Immuno-Cytokine Regulation in CyHV-3 Infection. | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 周晉澄 | |
dc.contributor.oralexamcommittee | 邱品文,涂堅 | |
dc.subject.keyword | 錦鯉?疹病毒,cyhv3IL-10,持續性感染,發炎反應,免疫逃脫, | zh_TW |
dc.subject.keyword | Cyprinid herpesvirus 3 (CyHV-3),cyhv3IL-10,persistent infection,inflammatory response,immunoescape?mechanisms, | en |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU201704253 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-10-12 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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