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研究生:林淑卿
研究生(外文):Lin Shu-Ching
論文名稱:研發水包油包水佐劑應用於新城病不活化疫苗
論文名稱(外文):Development of water-in-oil-in-water adjuvant applied Newcastle disease inactivated vaccine
指導教授:柯冠銘柯冠銘引用關係
指導教授(外文):Ke, Guan-Ming
口試委員:吳弘毅蔡信雄
口試委員(外文):Wu,Hung-YiTsai, Shin-shiung
口試日期:2016-06-28
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物疫苗科技研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:67
中文關鍵詞:水包油包水佐劑CpG-ODN新城雞病
外文關鍵詞:W/O/W adjuvantCpG-ODNNewcastle disease
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目錄
摘 要................................................. I
Abstract....................................... II
謝誌................................... IV
目錄................................... V
圖表目錄........................................ IX
第1章 緒言....................................... 1
第2章 文獻回顧................................... 3
2.1 新城病 (NEWCASTLE DISEASE; ND)................ 3
2.2 新城病與經濟影響.............................. 3
2.3 新城病病毒 (NEWCASTLE DISEASE VIRUS)......... 4
2.3.1 病毒結構................................... 4
2.3.2 NDV基因體.................................. 4
2.3.3 病毒的組合與釋放............................. 4
2.3.4 傳播途徑................................... 5
2.3.5 感染與臨床症狀.............................. 5
2.3.5.1 新城病防疫現況................................... 6
2.4 動物用疫苗佐劑.................................. 6
2.4.1 佐劑之分類................................. 7
2.4.2 佐劑之作用機制................................... 8
2.4.3 油質佐劑.................................... 9
2.4.4 乳劑....................................... 9
2.4.5 界面活性劑和HLB值.......................... 10
2.4.6 佐劑之應用................................ 10
2.4.7 CpG DNA 佐劑............................. 12
2.4.7.1 CpG motif 結構與分類.................... 13
2.4.7.2 CpG DNA之佐劑應用................ 14
第3章 材料與方法................................. 15
3.1 新城病病毒培養與力價測定................ 15
3.1.1 病毒增殖 ................................15
3.1.2 蛋胚半感染劑量測定(50% egg infectious dose, EID50) 15
3.1.3 雞血球懸浮液製備........................... 15
3.1.4 血球凝集試驗(Hemaggutination; HA)...... 16
3.1.5 新城病抗原不活化 ........................16
3.1.6 新城病抗原不活化完全測試................. 16
3.2 新城病毒分子生物學特性分析.................... 16
3.2.1 病毒RNA之萃取....................... 16
3.2.2反轉錄聚合酶連鎖反應 (Reverse transcription-PCR) 17
3.2.3聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 17
3.2.4 One-step反轉錄聚合酶連鎖反應(One-Step RT-PCR) 17
3.2.5 DNA膠體電泳法............................. 18
3.3 內毒素 (ENDOTOXIN) 含量檢測............ 18
3.4 新城病毒疫苗製備...................... 18
3.4.1 水包油包水佐劑製備................... 18
3.4.2 不活化疫苗之製備....................... 19
3.5 動物試驗 ................................19
3.5.1 最小致死劑量(Minimum lethal dose, MLD) 19
3.5.2 免疫計畫 ................................19
3.5.2.1 NDV-Sato疫苗安全試驗:................... 19
3.5.2.2 血清分離......................... 20
3.5.2.3 血球凝集抑制實驗 (Hemaggutanation inhibition test) 20
3.5.2.4 腫脹係數評估..................... 20
3.5.2.5 攻毒試驗......................... 21
第4章 結果 ................................22
4.1 NDV-SATO病毒株於蛋胚中生長情形.......... 22
4.2 EID50試驗檢測NDV-SATO經蛋胚培養後的病毒力價 22
4.3 HA試驗檢測NDV-SATO病毒力價............. 22
4.4 NDV基因片段確認...................... 22
4.5 內毒素(ENDOTOXIN)含量檢測........... 23
4.6 水包油包水調配最佳比例 ................23
4.7 效力試驗前攻毒株NDV最小致死劑量定量.......... 23
4.8 安全試驗 ................................24
4.9 血球凝集抑制試驗 ( HI ) ................24
4.10 腿圍測量 ................................24
4.11 攻毒試驗................................... 24
第5章 討論...................................... 38
參考文獻 ........................................42
作者簡介………………………………………………………………............... 67

圖表目錄
Table 1. Hemagglutination (HA) titer of NDV-Sato. ................................................26
Table 2. The formulation and stability of various emulsion....................................... 27
Table 3 MLD (minimum lethal dose) the smallest amount of NDV Sato (III) that will kill an experimental animal. .................................................28
Table 4 Virus shedding from cloacal swabs of vaccinated chickens after NDV-Sato challenge. .........29
Figure 1 chicken embryo (right) was inoculated with virus, causing whole body congestion and hemorrhage. .................................................30
Figure 2 Confirmation of NDV DNA. M: DNA molecular weight marker. lane 1: NDV-Sato DNA, lane 2: Allantoic fluid control group, lane 3-6: NDV (534 bp)、IBV (619 bp)、ILTV (797 bp)、AIV (330 bp) control group. The size of maker in kb is indicated on the left. ..................31
Figure 3 Photomicrograph of the developed W/O/W multiple emulsion immediately after preparation. (A) magnification 200× (B) magnification 400× ...................32
Figure 4 The Limulus amebocyte lysate test for endotoxin. (A) Positive control : E. coli endotoxin (B) Negative control : non-pyrogen water (C) The extract of CpG DNA. ...................................................33
Figure 5 Chickens vaccinated with the NDV-Sato used Ishii (III) strain for HI test. ...................34
Figure 6 The Leg swelling test of different test groups. (A) Control group around the average distribution of leg circumference (B) Immune groups around the average distribution of leg circumference. ............35
Figure 7 Anatomical Observation leg swelling. ....36
Figure 8 Lesions on visceral organs of chicken challenged with Sato (III) virus.......................... 37
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