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| 研究生： |
林育芬 Lin, Yu-Fen |
|---|---|
| 論文名稱： |
細胞培養雞傳染性支氣管炎疫苗之研發 Development of avian infectious bronchitis vaccine using cell culture production |
| 指導教授： |
柯冠銘
Ke, Guan-Ming |
| 學位類別： |
碩士 Master |
| 系所名稱： |
獸醫學院 - 動物疫苗科技研究所 Graduate Institute of Animal Vaccine Technology |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 84 |
| 中文關鍵詞： | 雞傳染性支氣管炎病毒 、交叉保護力 、Vero 細胞 、活毒疫苗 |
| 外文關鍵詞： | Avian infectious bronchitis virus, cross-protection, Vero cell, live vaccine |
| DOI URL： | http://doi.org/10.6346/NPUST201900114 |
| 相關次數： | 點閱：26 下載：0 |
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雞傳染性支氣管炎病毒(Avian infectious bronchitis virus；IBV)屬於冠 狀病毒屬之具封套膜單股正鏈 RNA 病毒。由於不同血清型間的 IBV 缺乏 交叉保護力，因此需要針對新的血清型 IBV 開發疫苗。IBV 可於雞胚胎 中增殖。但目前以雞胚胎增殖病毒株有 SPF 雞胚胎取得不易、成本過高 以及雞胚胎廢棄物處理之環保問題。因此本研究將建立培養 IBV 之合適 細胞系以生產病毒，目前利用 Vero 細胞培養 IBV 經 30 代馴化其病毒量 可達 106 TCID50 /mL，對照雞傳染性支氣管炎活毒疫苗之檢定標準，相當 於 107.3 EID50 /mL，細胞可以觀察到明顯的融合、空泡化與脫落死亡的 CPE 現象，以 103 TCID50 /mL 至 106 TCID50 /mL 之病毒量製備成活毒疫苗免疫 於雞隻，其 ELISA 測得之抗體力價於免疫後 2 至 3 週有明顯的上升，能 夠穩定維持至少 8 週，以血清中和試驗測得之抗體力價最高可達 8 倍， 其抗體可持續維持至免疫後第 10 週，綜合試驗結果，顯示具開發細胞培 養雞傳染性支氣管炎活毒疫苗之潛力。
Avian infectious bronchitis virus (IBV) belongs to coronavirus. It is an enveloped, single-stranded, positive-sense RNA virus. Because different serotypes of the virus don’t give cross-protection, it is necessary to develop a vaccine against the new serotype IBV. IBV can proliferate in chicken embryos. At present, however, it is not easy to obtain the embryo of the SPF chicken since the cost is too high. Waste treatment of chicken embryo is also problematic. Therefore, this study will establish a suitable cell line to produce IBV . For now, we have cultured and passaged IBV by using Vero cells and the current titers of IBV up to 106 TCID50 /mL after 30 generations of passage. Compared with the standard of avian infectious bronchitis live vaccine, it is equivalent to 107.3 EID50 /mL. The cells can observe the CPE phenomenon of obvious fusion, vacuolization, shedding and death. The IBV live vaccines were prepared with the virus amount of 103 TCID50 /mL to 106 TCID50 /mL to immunize chickens. The antibody titer measured by ELISA showed a significant increase from 2 to 3 weeks after immunization. It could be stably maintained for at least 8 weeks. The neutralization antibody titer could reach 8x dilutions, and the antibody could be maintained until the 10th week after immunization. The comprehensive test results show the potential to develop a cell-cultured infectious bronchitis vaccine.
中文摘要………………………………………………………………………I Abstract…………………………………………………………………II
謝誌…………………………………………………………………………IV
目錄…………………………………………………………………………V
圖表目錄…………………………………………………………………VIII
第1章 緒言…………………………………………………………………1
第2章 文獻回顧……………………………………………………………3
2.1 雞傳染性支氣管炎(Avian infectious bronchitis；IB)之簡介…………3
2.1.1 歷史背景…………………………………………………………3
2.1.2 流行病學…………………………………………………………5
2.1.3 感染機制…………………………………………………………6
2.1.4 臨床症狀…………………………………………………………7
2.1.5 病理變化…………………………………………………………8
2.1.6 免疫機轉…………………………………………………………9
2.1.7 疾病預防與控制…………………………………………………10
2.2 分子病毒學…………………………………………………………11
2.2.1 雞傳染性支氣管炎病毒之型態與特性…………………………11
2.2.2 雞傳染性支氣管炎病毒之結構與功能…………………………12
2.3 病毒之分類與分型…………………………………………………14
2.3.1 雞傳染性支氣管炎病毒之分類…………………………………14
2.3.2 雞傳染性支氣管炎病毒之血清型別與分型方法………………15 2.3.3 雞傳染性支氣管炎病毒之基因型別與鑑定方法………………16
2.4 病毒之診斷方法……………………………………………………17
2.4.1 雞傳染性支氣管炎病毒之分離…………………………………17
2.4.2 雞傳染性支氣管炎病毒之血清學診斷…………………………17
2.4.3 雞傳染性支氣管炎病毒之中和試驗……………………………18
2.4.4 即時定量反轉錄聚合酶連鎖反應(Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction ; qRT-PCR)…………19
2.5 疫苗研究歷史與發展………………………………………………19
第3章 材料與方法……………………………………………………21
3.1 病毒之來源與分離…………………………………………………21
3.1.1 雞傳染性支氣管炎病材及組織來源……………………………21
3.1.2 雞傳染性支氣管炎病毒株之分離………………………………21
3.2 病毒之純化與增殖…………………………………………………21
3.2.1 雞傳染性支氣管炎病毒之純化…………………………………21
3.2.2 雞傳染性支氣管炎病毒於SPF雞胚尿囊腔之增殖…………22 3.2.3 非洲綠猴腎細胞(Vero cell)之培養……………………………22 3.2.4 非洲綠猴腎細胞之冷凍保存…………………………………23
3.2.5 雞傳染性支氣管炎病毒於非洲綠猴腎細胞之培養…………23
3.3 病毒之檢測與鑑定…………………………………………………24
3.3.1 雞傳染性支氣管炎病毒核酸之萃取…………………………24
3.3.2 多重聚合酶連鎖反應(Multiplex Polymerase Chain Reaction)..25
3.3.3 雞傳染性支氣管炎病毒S1基因增幅之引子……………25
3.3.4 雞傳染性支氣管炎病毒S1基因之反轉錄聚合酶連鎖反應 (Reverse Transcription Polymerase Chain Reaction ; RT-PCR)………25
3.3.5 雞傳染性支氣管炎病毒S1基因之膠體電泳分析……………..26 3.3.6 雞傳染性支氣管炎病毒 S1 基因之 RT-PCR 產物之分子選殖..26
3.3.7 質體之純化………………………………………………………27
3.3.8 雞傳染性支氣管炎病毒 S1 基因之核苷酸序列比對與親緣關係 分析.……………………………………………………………………28
3.4 病毒之定量…………………………………………………………28
3.4.1 即時定量反轉錄聚合酶連鎖反應(Real-time Quantitative Reverse Transcription - Polymerase Chain Reaction ; qRT-PCR)………………28
3.4.2 雞傳染性支氣管炎病毒標準曲線之建立………………………28 3.4.3 雞胚胎半數感染劑量(medium embryo infective dose ; EID50)..29
3.4.4 組織培養半數感染劑量(50% tissue culture infectious dose ; TCID50)…………………………………………………………………29
3.4.5 雞傳染性支氣管炎病毒之TCID50與EID50力價之對比試驗…30 3.5 疫苗之製備….………………………………………………………30
3.5.1 活毒疫苗之製備…………………………………………………30
3.5.2 無菌試驗……………………………………………………….31
3.6 動物試驗……………………………………………………………31
3.6.1 試驗雞隻之物種來源……………………………………………31
3.6.2 疫苗安全性試驗…………………………………………………31
3.6.3 疫苗效力試驗及實驗動物組別設定……………………………31
3.6.4 血清分離…………………………………………………………32
3.7 血清抗體力價測定…………………………………………………32
3.8 酵素免疫分析法(Enzyme-linked immunosorbent assay ; ELISA).32
3.9 血清中和試驗………………………………………………………33
第4章 結果…………………………………………………………………34
4.1 多重聚合酶連鎖反應(Multiplex PCR)之野外病毒株純潔度試驗..34
4.2 雞傳染性支氣管炎病毒株於細胞中之馴化情形…………………34
4.3 反轉錄聚合酶連鎖反應(RT-PCR)檢測每代雞傳染性支氣管炎病 毒株於細胞中馴化之純潔度…………………………………………35
4.4 反轉錄聚合酶連鎖反應擴增雞傳染性支氣管炎病毒株之S1基因片 段…………………………………………………………………………35
4.5 雞傳染性支氣管炎病毒株之序列比對與親緣關係分析…………36 4.6 以TCID50試驗與即時定量反轉錄聚合酶連鎖反應(qRT-PCR)檢測 雞傳染性支氣管炎病毒經細胞培養後之病毒力價……………………36
4.7 雞傳染性支氣管炎病毒力價(TCID50)之即時定量反轉錄聚合酶連鎖反應(qRT-PCR)定量曲線………………………………………………36
4.8 雞傳染性支氣管炎病毒 TCID50 試驗與 EID50 試驗之病毒力價比對分析………………………………………………………………………37
4.9 動物試驗…………………………………………………………37
4.9.1 免疫血清之 ELISA 分析………………………………………37
4.9.2 免疫血清之中和抗體試驗………………………………………37
第5章 討論…………………………………………………………………57
參考文獻…………………………………………………………………61
附錄…………………………………………………………………………81
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