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| 研究生： |
林宥任 Yu-Jen Lin |
|---|---|
| 論文名稱： |
鴿子副黏液病毒不活化疫苗之研發 Development of Inactivated Vaccine Against Pigeon Paramyxovirus |
| 指導教授： |
柯冠銘
GUAN-MING KE |
| 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： | 110 |
| 語文別： | 中文 |
| 論文頁數： | 45 |
| 中文關鍵詞： | 鴿子副黏液病毒 、不活化疫苗 、佐劑 |
| 外文關鍵詞： | Pigeon Paramyxovirus, Adjuvant, Inactivated vaccine |
| DOI URL： | http://doi.org/10.6346/NPUST202200052 |
| 相關次數： | 點閱：49 下載：0 |
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鴿子副黏液病毒第一型(Pigeon paramyxovirus type 1;PPMV-1)在環境中傳播速度非常快且具有很強的傳染能力，會引起食慾不振、神經症狀及高死亡率，造成嚴重經濟損失。1978年首次在中東的肉鴿中發現了PPMV-1。之後在1980年代初期，該病毒隨之於歐洲和北美開始擴散，在許多飼主家中的賽鴿被檢測到，其中可能是經由飼料或糞便傳播。本研究目的是將研究室所分離鴿子副黏液病毒培養馴化，篩選低毒力、高抗原性種毒株並研發出不活化疫苗與制定出最佳免疫程序。馴化之PPMV-1藉由血球凝集 (Hemagglutination; HA)、TCID50以及EID50測定其病毒量，並搭配水油水或油質佐劑製成不活化疫苗，最後施打於鴿子中進行安全及效力之評估。本研究以BHK-21細胞培養PPMV-1經40代馴化其病毒量可達到108TCID50/mL，用SPF蛋培養其病毒量可達109EID50/mL，以兩種不同的培養方式培養之PPMV-1經福馬林不活化後與不同佐劑乳化，施打於鴿子進行效力、安定性之評估。結果顯示添加108EID50/mL與 Ishii搭配水油水佐劑製成不活化疫苗免疫鴿隻測定抗體力價，其抗體力價結果優於商用疫苗組，鴿隻攻毒後陰性對照組有排毒現象，而施打疫苗組別未從糞便中檢測到病毒，表示製備出的PPMV-1疫苗具有發展成商用疫苗之潛能。
Pigeon paramyxovirus type 1 (PPMV-1) spreads very fast in the environment and is strongly infectious. It causes loss of appetite, neurological symptoms, and high mortality resulted in serious economic losses. PPMV-1 was first discovered in meat pigeons in the Middle East in 1978. Later, in the early 1980s, the virus began to spread in Europe and North America. It was detected from racing pigeons in breeding environment, which may have been spread through feed or feces. In this study, the pigeon paramyxovirus was isolated in the laboratory, and then sub-cultured in BHK-21 cell. For attenuating virulence the vaccine was derived from low-virulence and high-antigenic strains. The viral load of domesticated was tested against Hemagglutination (HA), TCID50 and EID50. Two types of inactivated vaccine by formalin were emulsificated with water in oil in water and water in oil adjuvants, and then administered to pigeons for safety and efficacy evaluation. After 40 generations of domestication, production of PPMV-1 can reach 108TCID50/mL via culture in BHK-21 cells, and 109EID50/mL via culture in SPF eggs. This study showed the safety and efficacy evaluation of PPMV-1, which was cultured by two different methods, and emulsified using different adjuvant through administering to pigeons. The results showed that the group of 108EID50/mL PPMV-1 and Ishii with S550 adjuvant has a higher antibody titer in pigeons compared with commercial vaccine. After challenge, feces were detected virus in control group, not vaccinated group, indicating that the PPMV-1 vaccine has potential to be developed into commercial vaccine.
目錄
摘要 I
Abstract III
謝誌 V
目錄 VI
圖表目錄 IX
第1章 序言1
第2章 文獻回顧 3
2.1. 鴿子副黏液病毒第一型(Pigeon paramyxovirus type 1;PPMV-1)病毒介紹 3
2.2 PPMV-1流行病史 4
2.3 PPMV-1病毒結構組成 5
2.4 PPMV-1病毒蛋白特性 5
2.5 PPMV-1傳染途徑 5
2.6 PPMV-1感染症狀 6
2.7 PPMV-1預防與控制 6
2.8 PPMV-1鑑定方式 6
2.9 PPMV-1毒力測定 7
2.10 PPMV-1 F蛋白特性 8
2.11 F蛋白變異區 8
2.12 疫苗種類 9
2.13 滅活疫苗(Inactivated Vaccine) 10
2.14 甲醛(formaldehyde) 10
2.15 油質佐劑 11
2.16 水包油包水佐劑(Water-in-oil-in-water adjuvant) 12
第3章 材料與方法 13
3.1 巴拉米哥病毒增值和檢測力價 13
3.1.1 病毒於細胞增殖 13
3.1.2 病毒於胚胎蛋中增殖 13
3.1.3 血球凝集(Hemaggutination;HA)試驗 13
3.1.4 半數組織培養感染劑量 (50% Tissue culture infective dose;TCID50) 14
3.1.5 半數雞胚感染劑量(50% Embryo infective dose;EID50) 14
3.2 巴拉米哥病毒分子生物學特性分析 14
3.2.1 病毒RNA萃取 14
3.2.2 RNA病毒反轉錄 15
3.2.3 以PCR 擴增目標片段 15
3.2.4 膠體電泳確認產物大小與核酸序列定序 15
3.3 攻毒株之製備與病毒不活化 16
3.3.1 培養攻毒株 16
3.3.2 最小感染劑量（Minimal infective dose, MID）測定 16
3.3.3 病毒之不活化 16
3.3.3.1 TW-2018-08病毒不活化 16
3.3.3.2 新城雞病第七基因型(NDV VII)病毒不活化 16
3.3.3.3 新城雞病石井株(Ishii)病毒不活化 17
3.4 動物試驗設計與效力評估 17
3.4.1 第一次動物實驗 17
3.4.2 第二次動物實驗 17
3.4.3 第三次動物試驗 17
3.4.4 第四次動物實驗 18
3.4.5 血球凝集抑制(Hemaggutanation inhibition;HI)測試 18
3.4.6 攻毒試驗 18
3.5 排毒檢測 18
3.5.1 檢體採樣 18
3.5.2 即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction,qPCR) 19
第4章 結果 20
4.1 TW-2018-08病毒株繼代於BHK-21之生長情形 20
4.2 以TCID50與EID50檢測TW-2018-08於細胞與蛋培養的病毒力價 20
4.3 選殖巴拉米哥F蛋白基因片段 20
4.4 動物試驗與疫苗效力評估 20
4.4.1 免疫前攻毒株TW-2018-08定量 20
4.4.2 免疫前抗體力價檢測 21
4.4.3 第一次動物實驗-血清抗體反應 21
4.4.4 第二次動物實驗-血清抗體反應 21
4.4.5 第三次動物實驗-血清抗體反應 22
4.4.6 第四次動物實驗-血清抗體反應 22
4.4.7 免疫後攻毒 23
4.4.8 利用qPCR檢測攻毒後鴿隻排毒量 23
第5章 討論 36
第6章 參考文獻 39
圖表目錄
Table 1.Primers used for amplification of PPMV-1. 24
Table 2.Groups of pigeons vaccinated with S550 in the first animal test. 25
Table 3.Groups of pigeons vaccinated with various adjuvants in the second animal test. 26
Table 4.Groups of pigeons vaccinated with various adjuvants in the third animal test. 27
Table 5.Groups of pigeons vaccinated with various antigens in the fourth animal test. 28
Table 6.Virus shedding detection by qPCR. 29
Figure 1.The infected BHK-21 cells by TW-2018-08. 30
Figure 2.PCR amplification of F gene of PPMV-1 TW-2018-08. 31
Figure 3.HI titer response of the first animal trial. 32
Figure 4.HI titer response of the second animal trial. 33
Figure 5.HI titer response of the third animal trial. 34
Figure 6.HI titer response of the fourth animal trial. 35
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校外公開2027/01/26