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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉嚞睿(Zhe-Rui Liu) | |
| dc.contributor.author | Yu-Wei Zheng | en |
| dc.contributor.author | 鄭雨薇 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:39:12Z | - |
| dc.date.copyright | 2022-08-19 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85030 | - |
| dc.description.abstract | 豬繁殖與呼吸綜合症病毒 ( porcine reproductive and respiratory syndrome virus, PRRSV ) 的感染和傳播對全球養豬業構成嚴重威脅。無論是滅活疫苗還是減毒疫苗都不能有效預防和控制PRRSV的感染和傳播。因此，有必要拓寬新視野並製訂有效的預防策略。先前研究指出芽孢桿菌具有對哺乳動物病毒感染的抑制活性。然而，目前仍未有應用芽孢桿菌萃取物用於抗PRRSV活性相關報導。因此，本研究的目的是評估芽孢桿菌的體外抗PRRSV活性，包括貝萊斯芽孢桿菌AC ( Bacillus velezensis AC )、解澱粉芽孢桿菌LN ( Bacillus amyloliquefaciens LN ) 和地衣芽孢桿菌CK1 ( Bacillus licheniformis CK1 )。在評估芽孢桿菌菌株的體外抗病毒活性時，使用 LN、AC和CK1的細胞內提取物和細胞壁分別來測試它們的抗PRRSV活性。通過3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( MTT ) 測定法測定細胞活力，以證明芽孢桿菌提取物通過直接與病毒結合抑制病毒複製的作用。結果表明，LN、AC和CK1提取物通過抑制病毒貼附、進入有效地抑制了MARC-145細胞中PRRSV的感染，並且通過共培養病毒空斑測試，發現芽孢桿菌萃取物能有效降低病毒的效價，從而降低了病毒對細胞的感染。此外，通過處理芽孢桿菌萃取物後能有效降低因PRRSV 感染而引起的細胞中發炎細胞因子的 mRNA 表現量。總之，芽孢桿菌菌株LN、AC和CK1具有抑制PRRSV感染的潛力，並具有用作抗病毒飼料添加劑的潛力。 | zh_TW |
| dc.description.abstract | The infection and spread of the porcine reproductive and respiratory syndrome virus (PRRSV) has an important influence on the global pig industry. Neither inactivated nor attenuated vaccines can effectively prevent and reduce the infection and spread of PRRSV. There is a necessary need to investigate a new strategy to prevent the PRRSV infection. Previous reports have indicated the ability of probiotic Bacillus to lower the risk of vial infection response in mammals. However, the ability of anti-PRRSV infection with probiotic Bacillus was not reported. Therefore, the aim of this study was to evaluate the anti-PRRSV activities with Bacillus strains in vitro, including Bacillus velezensis AC, Bacillus amyloliquefaciens LN, and Bacillus licheniformis CK1. In the evaluation of antiviral activity of Bacillus strains, the intracellular extracts and cell wall fractions of LN, AC and CK1 from Bacillus were used to measure their anti-PRRSV activity. The cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for the effect of Bacillus extracts on inhibition of viral replication, through directly binding to the virus, internalization, or inhibitory of viral replication. The results showed that LN, AC, CK1 extracts effectively inhibited PRRSV infection in MARC-145 cells by suppressing the viral binding and internalization. On the other hand, the co-culture virus plaque test indicated that the Bacillus extract can effectively reduce the titer and the ability of infection with PRRSV. Moreover, the mRNA expression levels of the pro-inflammatory cytokines in PRRSV-infected MARC-145 cells were decreased by the addition of Bacillus strains LN, AC, and CK1. In conclusion, Bacillus strains LN, AC, and CK1 may have the ability to inhibit PRRSV infection and can be used as an antiviral feed additive. | en |
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| dc.description.tableofcontents | 致謝 i 中文摘要 iii Abstract iv 目錄 v 圖目錄 ix 表目錄 xi 序言 1 第一章、 文獻回顧 2 第一節、 益生菌 2 一、 益生菌之定義與歷史 2 二、 益生菌菌株選擇 2 （一） 安全性 3 （二） 功能性 3 （三） 技術性 3 三、益生菌功能 3 （一）改善腸道黏膜屏障 4 （二）分泌有益功能性物質 4 （三）調節免疫系統 4 （四）抗氧化活性 5 第二節、 芽孢桿菌 11 一、 芽孢桿菌簡介 11 二、 贝莱斯芽孢桿菌 ( Bacillus velezensis ) 11 三、 地衣芽孢桿菌 ( Bacillus licheniformis ) 12 四、 液化澱粉芽孢桿菌 ( Bacillus amyloliquefaciens ) 12 第三節、 病毒 15 一、 病毒定義 15 二、 常見經濟動物病毒性疾病 15 （一）豬環狀病毒 ( Porcine circovirus, PCV ) 15 （二）豬流行性下痢 ( Porcine epidemic diarrhea, PED ) 15 （三）豬繁殖與呼吸綜合症 ( Porcine reproductive and respiratory syndrome, PRRS ) 16 1. PRRSV形態特徵 16 2. PRRSV基因組特徵 16 3. PRRSV複製週期 17 4. PRRSV感染階段 17 5. PRRSV臨床特徵及其防治 18 第四節、 研究動機及目的 24 第二章、 材料與方法 25 第一節、 實驗框架 25 第二節、 菌株、細胞株、病毒培養 26 一、 芽孢桿菌培養與保存 26 二、 芽孢桿菌萃取物製備 26 （一）胞內萃取物 ( Intracellular extract ) 26 （二）細胞壁 ( Cell wall ) 27 三、 細胞株培養 27 （一）細胞培養液製備 27 （二）細胞株活化 27 （三）細胞株繼代 27 （四）細胞株冷凍保存 28 四、 豬繁殖與呼吸綜合症病毒之培養 28 （一）病毒感染液製備 28 （二）病毒製備 28 （三）病毒感染力測試 29 （四）病毒效價測定 29 第三節、芽孢桿菌萃取物體外抗豬繁殖與呼吸綜合症病毒評估 30 一、細胞毒性測試 30 （一）MTT製備 30 （二）芽孢桿菌萃取物細胞毒性測試 30 二、預防模式下抗病毒活性測試 31 三、 治療模式下抗病毒活性測試 32 四、 共培養模式下抗病毒活性測試 32 （一） 共培養下直接抑制病毒 32 （二）共培養下抑制病毒貼附 33 （三）共培養下抑制病毒進入 33 （四）共培養下抑制病毒複製 34 五、 抗病毒及發炎相關基因表現 34 （一）抽取細胞中RNA 35 （二）抽取病毒RNA 35 （三）製備cDNA 36 （四）病毒標準品製備 36 （五）即時定量聚合酶連鎖反應 37 六、 共培養模式下病毒空斑測試 38 七、 芽孢桿菌萃取物與病毒直接作用 38 八、 統計分析 39 第三章、 實驗結果 45 第一節、顯微鏡下細胞形態和病毒感染情形 45 一、 正常生長下細胞形態 45 二、 細胞感染情況 45 第二節、豬繁殖與呼吸綜合症病毒毒性測定 45 一、 豬繁殖與呼吸綜合症病毒效價測試 45 第三節、 芽孢桿菌萃取物之體外抗豬繁殖與呼吸綜合症病毒能力評估 46 一、 菌株萃取物之細胞毒性 46 二、 預防模式下抗病毒活性 47 三、 治療模式下抗病毒活性 47 四、 共培養模式下抗病毒活性 48 （一）共培養模式下細胞活性測試 48 1. 共培養下直接抑制病毒 48 2. 共培養下抑制病毒貼附 49 3. 共培養下抑制病毒進入 49 4. 共培養下抑制病毒複製 49 （二）基因表現 49 1． 抗病毒基因分析 50 2． 發炎因子基因分析 51 3． 細胞內外病毒表現量 52 （三）共培養模式下病毒空斑測試 52 （四）共培養模式下直接抑制病毒 52 第四章、討論 88 第五章、結論 92 第六章、參考文獻 93 圖目錄 圖 1-1、菌株選擇標準 10 圖 1-2、豬繁殖與呼吸綜合症病毒粒子的冷凍電子顯微鏡圖像 19 圖 1-3、豬繁殖與呼吸綜合症病毒基因組、轉錄和轉譯 19 圖 1-4、豬繁殖與呼吸綜合症病毒複製週期 20 圖 1-5、豬繁殖與呼吸綜合症病毒感染之免疫反應 21 圖 1-6、I型干擾素誘導下的抗病毒活性 22 圖 2-1、實驗架構 24 圖 2-2、豬繁殖與呼吸綜合症病毒不同稀釋濃度下對MARC-145細胞存活率測定之時間流程圖 41 圖 2-3、芽孢桿菌萃取物之細胞毒性實驗流程圖 41 圖 2-4、預防模式下抗豬繁殖與呼吸綜合症病毒之實驗流程 41 圖 2-5、治療模式下抗豬繁殖與呼吸綜合症病毒之實驗流程 42 圖 2-6、共培養模式下抑制豬繁殖與呼吸綜合症病毒之實驗流程及RNA萃取時間點 42 圖 2-7、共培養模式下抑制豬繁殖與呼吸綜合症病毒貼附之時間流程圖 42 圖 2-8、共培養模式下抑制豬繁殖與呼吸綜合症病毒進入之時間流程圖 43 圖 2-9、共培養模式下抑制豬繁殖與呼吸綜合症病毒複製之時間流程圖 43 圖 2-10、豬繁殖與呼吸綜合症病毒標準品定序結果 44 圖 3-1、顯微鏡觀察下細胞正常生長狀況下長至單層情形 53 圖 3-2、不同時間點正常細胞和受感染細胞細胞形態和凋亡程度的變化 54 圖 3-3、病毒原液序列稀釋下病毒空斑實驗 55 圖 3-4、豬呼吸與繁殖綜合症病毒在不同稀釋倍數下對MARC-145細胞的存活率測定 56 圖 3-5、芽孢桿菌萃取物對MARC-145細胞的細胞毒性測定 57 圖 3-6、預防模式下芽孢桿菌萃取物對MARC-145細胞的抗PRRSV活性測定 58 圖 3-7、治療模式下芽孢桿菌萃取物對MARC-145細胞的抗PRRSV活性測定 60 圖 3-8、共培养模式下芽孢桿菌萃取物對MARC-145細胞的抗PRRSV活性測 62 圖 3-9、共培养模式下芽孢桿菌萃取物對MARC-145細胞的存活率影響 64 圖 3-10、共培養模式下抑制病毒貼附 65 圖 3-11、共培養模式下抑制病毒進入 67 圖 3-12、共培養模式下抑制病毒複製 69 圖 3-13、芽孢桿菌萃取物對I型干擾素相關抗病毒基因表現的影響 71 圖 3-14、芽孢桿菌萃取物對促發炎細胞激素基因的影響 75 圖 3-15、芽孢桿菌萃取物於共培養處理下對細胞內外PRRSV-M拷貝數的影響 81 圖 3-16、芽孢桿菌萃取物於共培養模式下抑制病毒空斑測試 83 圖 3-17、芽孢桿菌萃取物於不同時間點處理下之病毒包膜蛋白表現 87 表目錄 表 1-1、益生菌之定義 6 表 1-2、主要用於動物的益生菌菌株 8 表 1-3、文獻記載用於動物和人類之芽孢桿菌 14 表 2-1、即時定量聚合酶連鎖反應之引子序列及其反應條件 40 表 3-1、發炎基因水平表現分析表 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 益生菌 | zh_TW |
| dc.subject | 芽孢桿菌 | zh_TW |
| dc.subject | 抗病毒 | zh_TW |
| dc.subject | 體外 | zh_TW |
| dc.subject | 豬繁殖與呼吸綜合症 | zh_TW |
| dc.subject | Anti-viral | en |
| dc.subject | Probiotic | en |
| dc.subject | Bacillus | en |
| dc.subject | porcine reproductive and respiratory syndrome virus | en |
| dc.subject | in vitro | en |
| dc.title | 具益生菌特性之芽孢桿菌的抗病毒活性評估 | zh_TW |
| dc.title | Evaluation of antiviral activities of Bacillus strains with probiotic characteristics | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭永祥(Yong-Xiang Zheng),張惠雯(Hui-Wen Zhang),劉啟德(Qi-De Liu) | |
| dc.subject.keyword | 益生菌,芽孢桿菌,豬繁殖與呼吸綜合症,體外,抗病毒, | zh_TW |
| dc.subject.keyword | Probiotic,Bacillus,porcine reproductive and respiratory syndrome virus,in vitro,Anti-viral, | en |
| dc.relation.page | 100 | |
| dc.identifier.doi | 10.6342/NTU202202452 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-08-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物科技研究所 | zh_TW |
| dc.date.embargo-lift | 2022-08-19 | - |
| 顯示於系所單位： | 生物科技研究所 | |
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