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研究生:江文瑀
研究生(外文):Wen-Yu Chiang
論文名稱:羊接觸傳染性化膿性口炎病毒不活化疫苗與檢測試劑開發
論文名稱(外文):Development the inactivated vaccine and diagnosis ELISA kit for orf virus
指導教授:莊國賓
指導教授(外文):Kuo-Pin Chuang
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:2013
畢業學年度:101
語文別:中文
論文頁數:88
中文關鍵詞:羊接觸傳染性化膿性口炎ORFVB2L不活化疫苗
外文關鍵詞:Contagious pustular dermatitisOrf virusrB2LInactivated vaccine
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羊接觸傳染性化膿性口炎(Orf)病毒為副痘病毒屬具有套膜的雙股DNA病毒,主要流行於畜牧業發達的國家。羊接觸傳染性化膿性口炎病毒為親上皮性,會造成嘴巴紅斑、丘疹、水泡、膿疱和結痂,可由飛沫傳播、昆蟲叮咬或直接接觸含有病毒之皮屑、分泌物、糞尿或是吸入含病毒之粉塵等傳播,主要宿主為羊隻,人則因接觸受感染的羊隻而意外感染,傳染率將近100%,所以有必要開發羊接觸傳染性化膿性口炎的疫苗與檢測試劑。我們用細胞培養方式增殖ORFV,並用陽性羊隻血清做ELISA確認所培養出的ORFV具有抗原性,在電子顯微鏡下也可觀察到明顯的病毒顆粒。另一方面,我們用PCR方式增殖B2L基因裝載入pET28a載體表現B2L蛋白,並用Anti-HIS與陽性羊隻血清確認B2L蛋白具有抗原性。將表現出的蛋白製作成ELISA檢測試劑和與不活化的Orf疫苗免疫動物並評估功效,我們先用小鼠做為動物實驗之前試驗,再由其中選擇三組施打於羊隻。在ELISA抗體偵測方面,小鼠實驗結果顯示只免疫不活化病毒與次單位蛋白之組別都無法有效的提升老鼠的抗體反應,但將不活化病毒與次單位蛋白混合施打之實驗組與控制組相比較在第14天時則有兩倍以上的抗體上升反應;而在羊隻實驗也是在混合施打之實驗組與控制組相比在第21天時有兩倍以上的抗體上升反應。在中和性抗體方面,小鼠實驗結果顯示,混合施打之組別在第14天時其中和能力高於控制組四倍。我們希望檢測試劑與疫苗在未來可對疾病的預防有所幫助。
Orf virus is the genus Parapoxvirus with envelope and double-strains DNA of. Orf mainly occurred in the animal husbandry developed country. Orf is an epitheliotropic. It caused erythema, papules, vesicles, pustules, and scab around the muzzle. Orf spread with small drops of contaminated water, insect bites or direct contact of virus dander, secretions, feces or inhalation of dust containing the virus. The main hosts are the sheep and goats, accidentally human were infected by contact with infected sheep and goats. Orf has very high morbidity up to 100%, so diagnosis kit and vaccine are very important to prevent virus transmission. Nowadays, no diagnostic kit is used in Taiwan. So we amplified the rB2L gene by PCR and cloned into pET28a vectors for protein expression. The antigenicity of rB2L protein was confirmed and used for the ELISA diagnostic kit development. On the other hand, we established the cell culture system for orf virus production and confirmed the virus antigenicity by ELISA with orf infected goat serum. The rB2L protein and inactivated orf virus were tested their immunogenicity using mouse and goat model. We found that vaccinated with inactivated orf virus plus rB2L had significantly higher neutralizing abtibody titer and Th1 cytokines response than those vaccinated with inactivated orf virus or rB2L only. According to these results, we hopes the diagnostic kit and vaccine will be helpful to disease prevention in the near future.
中文摘要 I
Abstract III
謝 誌 V
目 錄 VI
圖表目錄 X
第一章 序言 1
第二章 文獻回顧 3
2.1 羊接觸傳染性化膿性口炎之歷史背景 3
2.2 羊接觸傳染性化膿性口炎之宿主範圍 3
2.3 羊接觸傳染性化膿性口炎病毒之流行病學 4
2.4 羊接觸傳染性化膿性口炎病毒之特性 4
2.4.1病毒分類 4
2.4.2病毒型態與構造 5
2.5 羊接觸傳染性化膿性口炎病毒蛋白之功能特性 5
2.6 羊接觸傳染性化膿性口炎之感染途徑及致病機轉 6
2.7 羊接觸傳染性化膿性口炎之臨床症狀及病理學變化 7
2.8 羊接觸傳染性化膿性口炎之診斷 8
2.8.1 觀察組織病變 9
2.8.2 電子顯微鏡 9
2.8.3 血清學試驗 9
2.8.4 病毒分離 9
2.8.5 聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 10
2.9 羊接觸傳染性化膿性口炎之防治 11
2.10 羊接觸傳染性化膿性口炎之疫苗發展及現況 11
2.11 羊接觸傳染性化膿性口炎與羊痘之差異 12
2.12 原核pET表現系統 12
2.13 研究目的與動機 13
第三章 材料與方法 14
3.1 實驗方法與架構 14
3.2 病材來源 16
3.3 病毒診斷用引子對 16
3.4 ORFV之B2L蛋白表現 17
3.4.1病毒核酸萃取 17
3.4.2 聚合酶鏈鎖反應 17
3.4.3 DNA電泳分析 18
3.4.4 PCR產物純化回收 18
3.4.5 接合作用 19
3.4.6 勝任細胞之製備 19
3.4.7 轉形作用 19
3.4.8 菌落之篩選 20
3.4.9 小量質體DNA之萃取與純化 20
3.4.10 細菌的保存 20
3.4.11 核酸之定序與分析 21
3.4.12 PCR產物裝載pET28a載體並確認 21
3.4.13蛋白質誘導 21
3.4.14 蛋白質萃取 22
3.4.15 SDS-PAGE蛋白質電泳 22
3.4.16 西方轉漬分析及冷光壓片 23
3.4.17 蛋白質之純化 24
3.4.18 ELISA檢測試劑之抗原抗體定量 25
3.4.19 ELISA 26
3.5 不活化疫苗之製備 26
3.5.1 細胞株 26
3.5.2 細胞的解凍 26
3.5.3 細胞的繼代培養 27
3.5.4 細胞的冷凍保存 27
3.5.5 ORFV病毒培養 28
3.5.6 利用PCR方式確認ORFV病毒之DNA 28
3.5.7 ORFV病毒增殖 28
3.5.8 ORFV病毒純化 28
3.5.9 病毒定量 29
3.5.10 不活化病毒 29
3.6 實驗動物設計流程 30
3.6.1 實驗動物物種來源 30
3.6.2 實驗動物組別設定 30
3.7免疫效力試驗 32
3.7.1抗體測定 32
3.7.2血清中和試驗 32
3.7.3血液分離 32
3.7.4 T細胞增生試驗 33
3.7.5 RNA萃取 33
3.7.6轉cDNA 34
3.7.7細胞激素測定 34
第四章 結果 36
4.1 經由原核表現系統生產ORFV-B2L次單位蛋白 36
4.1.1 增幅ORFV-B2L目標基因全長 36
4.1.2 確認ORFV-B2L全長並裝載入yT&;A載體 36
4.1.3 確認ORFV-B2L全長並裝載入pET28a載體 36
4.1.4 製備ORFV-B2L之重組蛋白 37
4.1.5 利用SDS-PAGE蛋白質電泳分析經IPTG誘導之結果 37
4.1.6 利用西方墨點法確認ORFV-B2L蛋白 37
4.1.7 用SDS-PAGE來分析經由不同濃度的imidazol純化ORFV-B2L蛋白之結果 38
4.1.8 ORFV-B2L蛋白純化後用SDS-PAGE與Western blot分析之結果 38
4.1.9 利用西方墨點法確認ORFV-B2L蛋白之抗原性 38
4.1.10 利用ELISA確認ORFV-B2L蛋白的濃度與血清稀釋倍數之最佳條件 38
4.1.11 利用自製的ELISA檢測試劑進行樣本測試 39
4.2 ORFV的培養與不活化病毒的開發 39
4.2.1 利用PCR檢測野外羊隻結果 39
4.2.2 利用細胞培養來增殖ORFV 39
4.2.3 利用PCR來確認ORFV之增殖 40
4.2.4 ORFV之純化與濃縮 40
4.3 動物實驗 40
4.3.1 免疫不活化ORFV與rB2L次單位蛋白之小鼠血清抗體偵測 40
4.3.2 免疫不活化ORFV與rB2L次單位蛋白之小鼠血清中和性抗體 40
4.3.3 免疫不活化ORFV與rB2L次單位蛋白之羊隻血清抗體偵測 41
4.3.4 免疫不活化ORFV與rB2L次單位蛋白之羊隻血清中和性抗體 41
4.3.5免疫不活化ORFV與rB2L次單位蛋白之羊隻細胞激素測定 41
第五章 討論 63
第六章 參考文獻 68
附 錄 75
作者簡介 77

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