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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王金和(Ching-Ho Wang) | |
dc.contributor.author | Chien-Hao Chen | en |
dc.contributor.author | 陳建豪 | zh_TW |
dc.date.accessioned | 2021-06-13T05:47:10Z | - |
dc.date.available | 2007-07-27 | |
dc.date.copyright | 2006-07-27 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-10 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33838 | - |
dc.description.abstract | 1986年至2003年間台灣陸續有分離到H6N1、H7N1等弱毒型家禽流行性感冒病毒 (AIV),在2004年間計有22間養禽場之38萬隻雞因發現弱毒H5N2 AIV潛藏而遭致撲殺。此表示目前台灣已有H5和H6亞型禽流感感染的情形,為瞭解目前於台灣廣泛流行之H5N2和H6N1 AIV是否會經由雞胚胎繼代而改變其HA力價,並且觀察病毒基因與毒力是否有改變,本實驗將家畜衛生試驗所分讓野外病毒3233 (H5N2) 與2838D (H6N1) 毒株分別以SPF雞胚胎尿囊腔連續繼代至45代,選殖出初代與45代之HA1基因進行其序列分析比較,結果僅於3233 (H5N2)毒株第45代之EID50較初代上升約100倍。在HA1基因序列比對分析繼代前後H5有2個胺基酸變異。在HA蛋白表現方面,本實驗也從野外病毒株H5N2與H6N1針對HA基因序列上誘導產生中和抗體區域進行選殖,並分別構築於原核表現系統載體pGEX-5X-1及真核表現載體pcDNA3.1載體上進行蛋白質的表現,以發展次單位疫苗,將原核表現載體表現出的蛋白進行純化後以皮下肌肉注射方式免疫接種於4週齡SPF雞隻,2週後進行補強注射,在免疫及補強注射後2週採其血液以血球凝集抑制試驗檢測H6亞型HI抗體力價呈陽性 (HI抗體力價為24~26),而H5亞型HI抗體力價則呈陰性。另外,為暸解微脂體複合物是否可協同疫苗而增強免疫效力,本試驗利用市售新城病疫苗以微脂體與幾丁質進行包覆製成複合型疫苗,免疫接種實驗雞隻檢測其新城雞病HI抗體力價,實驗結果顯示疫苗以微脂體和幾丁質包覆後所產生的免疫效果比單獨以微脂體包覆的疫苗來的好。 | zh_TW |
dc.description.abstract | From 1986 to 2003, the avian influenza viruses (AIV) isolated in Taiwan were all low pathogenic. Some low pathogenic H5N2 AIVs were isolated in Taiwan in 2004, resulting in stamping 380 thousands chickens out from 22 poultry flocks. To test if the virus titers increased during passaging, H5N2 and H6N1 isolates were passaged in specific-pathogen-free embryonated eggs. After 45 passages, the virus titer of H5N2 increased 100 times, and two amino acid residues in HA1 of H5N2 virus were changed. The partial fragments of HA1 relating to neutralizing epitopes of the H5N2 and the H6N1 isolates were cloned in pGEX-5X-1 vector (prokaryotic expression) and pcDNA3.1 vector (eukaryotic expression) for expression. The purified recombinant proteins were intramuscualarly injected in 4-week-old SPF chickens for two times. Two weeks after the second immunization, the serum from H6-immunized chickens showed hemagglutination inhibition (HI) titers of 24 to 26, but no antibody was detected in H5-immunized chickens. To test the increase efficiency of liposome compounds on vaccine, Newcastle disease vaccines coated with liposome and with liposome/chitosan were used to immunize chickens. The results showed that Newcastle disease vaccine coated with liposome/chitosan increased HI titers in vaccinated chickens but not vaccine coated with lipsome alone. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T05:47:10Z (GMT). No. of bitstreams: 1 ntu-95-R93629020-1.pdf: 3898512 bytes, checksum: 2cc37ffb38fe4ede02b73915fbb5d1c8 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目 錄.....................................................I
表 次....................................................VI 圖 次...................................................VII 摘 要....................................................IX Abstract..................................................X 第一章 序言..............................................1 第二章 文獻探討..........................................2 第一節 家禽流行性感冒病毒歷史背景與流行病學..............2 第二節 家禽流行性感冒病毒性狀............................6 2.1 病毒特性..............................................6 2.2 家禽流行性感冒病毒基因體及蛋白質結構..................7 2.3 家禽流行性感冒病毒的複製機制.........................12 2.4 家禽流行性感冒病毒抗原變異的特性.....................13 2.5 家禽流行性感冒病毒物理與化學特性.....................14 第三節 家禽流行性感冒病毒之感染宿主範圍與致病機制.......15 3.1 感染自然宿主範圍.....................................15 3.2 家禽流行性感冒病毒致病機制...........................16 3.3 宿主感染家禽流行性感冒病毒後的免疫反應...............18 3.4 家禽流行性感冒病毒疫苗發展及防疫計劃.................18 第四節 家禽流行性感冒病毒之實驗室診斷方法...............20 4.1 病原直接分離.........................................20 4.2 檢測病毒抗原.........................................21 4.3 檢測病毒抗體.........................................22 第五節 家禽流行性感冒病毒病原性評估.....................23 5.1 病原性之評估.........................................23 5.2 高病原性家禽流行性感冒病毒之判定.....................24 第六節 微脂體在基因治療與疫苗發展的情形.................26 6.1 微脂體(liposome)...................................26 6.2 影響微脂體穩定性之生理因素...........................27 第三章 材料與方法........................................28 第一節 台灣家禽流行性感冒病毒H5、H6亞型毒株實驗室繼代與HA1序列分析.................................................28 3-1.1 病毒株來源........................................28 3-1.2 病毒繼代增殖......................................28 3-1.3 雞紅血球懸浮液的製備..............................29 3-1.4 血球凝集試驗(hemagglutination test;HA test)....29 3-1.5 50%胚胎蛋感染劑量(mean embryo infective dose;EID50)..................................................30 3-1.6 病毒核酸之萃取....................................30 3-1.7 增幅HA1基因引子...................................31 3-1.8 反轉錄聚合酶鏈鎖反應..............................31 3-1.9 瓊脂醣凝膠電泳(agarose gel electrophoresis).....32 3-1.10 病毒核酸反轉錄聚合酶鏈反應產物之純化.............33 3-1.11 TA-cloning kit 進行病毒核酸HA1基因之選殖(cloning)..............................................33 3-1.12 HA1選殖基因核苷酸定序............................35 3-1.13 HA1基因序列分析..................................35 第二節 微脂體包覆型疫苗發展與效力分析....................36 3-2.1 疫苗來源..........................................36 3-2.2 實驗動物..........................................36 3-2.3 實驗動物飼料與飲水................................36 3-2.4 雞新城病活毒疫苗病毒株之力價測定..................36 3-2.5 包覆型新城雞病疫苗微脂粒製備......................37 3-2.6 微脂體包覆型疫苗免疫效力試驗......................37 3-2.7 血球凝集抑制試驗hemagglutiniation inhibition test(HI test)..............................................38 第三節 家禽流行性感冒病毒HA1蛋白表現及確認...............38 3-3.1 家禽流行性感冒病毒HA1基因引子設計.................39 3-3.2 以反轉錄聚合酶鏈反應增幅HA1基因...................41 3-3.3 反轉錄聚合酶鏈反應產物之確認......................41 3-3.4 病毒HA1 基因反轉錄聚合酶鏈反應產物之純化..........41 3-3.5 表現載體之製備....................................42 3-3.6 病毒HA1表現基因之製備.............................42 3-3.7 表現載體之接合反應(ligation)....................42 3-3.8 表現重組載體之轉型作用(transformation)..........42 3-3.9 表現重組載體之確認................................43 3-3.10 表現重組載體之抽取................................44 3-3.11 表現重組載體DNA之限制酶切割確認..................44 3-3.12 原核表現系統勝任細胞之製備.......................44 3-3.13 原核重組蛋白之表現...............................45 3-3.14 原核重組蛋白之純化...............................46 3-3.15 原核重組蛋白之電泳分析與確認.....................46 3-3.16 西方墨點法確認重組蛋白之特異性...................47 3-3.17 原核重組蛋白之定量...............................47 3-3.18 真核表現載體表現細胞之培養.......................48 3-3.19 真核表現載體之轉染作用(transfection)...........48 第四節 重組蛋白之應用....................................49 3-4.1 原核蛋白油質乳劑疫苗之製備........................49 3-4.2 原核重組蛋白之動物試驗............................50 第四章 結果..............................................51 第一節 台灣家禽流行性感冒病毒H5、H6亞型毒株實驗室繼代與HA1序列分析.................................................51 4-1.1 H5、H6亞型毒株實驗室繼代之HA力價..................51 4-1.2 H5、H6亞型毒株繼代前後病毒毒力分析................51 4-1.3 H5、H6亞型毒株繼代前後其HA1基因序列分析...........51 第二節 微脂體包覆型疫苗效力分析.........................52 4-2.1 NDV疫苗病毒株的力價測定...........................52 4-2.2 SPF雞隻接種微脂體包覆型疫苗HI力價之檢測...........52 第三節 家禽流行性感冒病毒HA1蛋白表現及確認...............53 4-3.1 原核重組質體的確認................................53 4-3.2 真核重組質體的確認................................53 4-3.3 原核重組蛋白之表現................................54 4-3.4 原核重組蛋白之純化................................54 4-3.5 原核重組蛋白之西方墨點法(western blotting)......54 4-3.6 原核重組蛋白質之定量..............................55 4-3.7 真核重組蛋白之表現................................55 4-3.8 真核重組蛋白之西方墨點法(western blotting)......55 第四節 重組蛋白之應用....................................56 4-4.1 原核重組蛋白之動物試驗............................56 第五章 討論..............................................57 第一節 台灣家禽流行性感冒病毒H5、H6亞型毒株實驗室繼代與HA1序列分析.................................................57 第二節 微脂體包覆型疫苗效力分析..........................60 第三節 家禽流行性感冒病毒HA1蛋白表現及確認...............61 第四節 重組蛋白之應用....................................63 參考文獻.................................................85 | |
dc.language.iso | zh-TW | |
dc.title | 家禽流行性感冒病毒血球凝集素蛋白之表現及其應用 | zh_TW |
dc.title | Expression and application of avian influenza virus hemagglutinin protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林茂勇,連一洋(Yi-Yang Lien),蔡向榮 | |
dc.subject.keyword | 家禽流行性感冒病毒,血球凝集素蛋白,疫苗, | zh_TW |
dc.subject.keyword | Avian influenza virus,Hemagglutinin protein,Vaccine, | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-12 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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