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研究生:吳靜如
研究生(外文):JingRu Wu
論文名稱:應用大腸桿菌表現家禽流行性感冒病毒血球凝集素以區別病毒血清亞型
論文名稱(外文):Application of Escherichia coli-Expressed Hemagglutinin of Avian Influenza Virus in Viral Serotyping
指導教授:張伯俊
指導教授(外文):PoaChun Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:2001
畢業學年度:89
語文別:中文
論文頁數:76
中文關鍵詞:家禽流行性感冒血球凝集素血清亞型
外文關鍵詞:Avian influenza (AI)Hemagglutinin (HA)Serotyping
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家禽流行性感冒 (Avian Influenza, AI),係由A型流行性感冒病毒所引起全身性或呼吸道感染的疾病。在禽類已證實有15種HA血清亞型 (HA1-HA15) 及9種NA血清亞型 (NA1-NA9),且水禽類為所有HA1-HA15亞型AI病毒之貯留者 (reservoir)。1997年在香港爆發H5N1強毒型AI大流行,此H5N1病毒對雞群具有高致病性,並可直接感染人類造成死亡,此一發現更加突顯AI病毒可藉基因重組,而形成新的高致病性病毒株之重大隱憂。目前最常用以區分AI病毒血清亞型之方法為血球凝集抑制試驗 (hemagglutination inhibition test, HI),此方法缺乏敏感性及快速性。本研究利用反轉錄酶鏈反應 (Reverse transcription polymerase chain reaction, RT-PCR),增幅HA1~HA15亞型病毒之HA基因片段,並將這些片段分別嵌入pET-32a表現載體內,再轉型至大腸桿菌BL-21 (DE3)中,進行重組HA蛋白質表現。這些重組蛋白經純化後,以西方轉印分析法 (Western blot analysis) 證實,HA1~HA15亞型之重組蛋白,對於15種HA血清亞型之雞隻高免血清具有極高之特異性,即單一亞型之重組蛋白僅能被相同亞型之高免血清所辨識,因此利用這些重組蛋白進行Western blot,可快速區分病毒抗血清之亞型。此外,HA3、HA5、HA6、HA7、HA9亞型之重組蛋白,亦被應用於塗鍍 (coating) ELISA plate,製備ELISA套組作為診斷用途。結果發現,此ELISA套組則可區分出HA3、HA5、HA6、HA7、HA9抗血清之亞型。另外,以這些重組蛋白進行Western blot或ELISA分析,亦成功診斷出台灣田間雞隻被HA6亞型病毒感染之案例。本研究之結果證實以原核系統所表現之HA重組蛋白,可用於區分AI病毒抗血清之亞型,此為一新的發現,將可用於協助改進現有之診斷技術,以達快速診斷AI病毒並區分其亞型之需求。
Avian influenza (AI) virus, a type A influenza virus, which might cause from a local respiratory to a systemic fatal infection in the affected animals. Fifteen HA subtypes (HA1~HA15) and nine NA subtypes (NA1~NA9) of avian influenza virus (AIV) have been found in avian species, and waterfowls are the main reservoir of these viruses. An outbreak of H5N1 virus infection occurred in Hong Kong in 1997; the virus was found highly pathogenic to chickens, and caused a lethal infection in human. This finding protrudes the importance of the generation of highly pathogenic viruses by recombination between different AIVs. The conventional method for differentiating subtypes of AIV is hemagglutination inhibition (HI) test, which is both time-consuming and insensitive. In this study, the HA gene fragment of 15 subtypes of AIV were amplifies by RT-PCR, and cloned individually into the expression vector pET32a. The recombinant plasmids were transformed into E. coli host BL-21 (DE3) for expression of recombinant HA proteins. After purification, these recombinant proteins were found to be useful in Western blot analysis for the differentiation of HA1~HA15 antisera. Moreover, the recombinant HA3, HA5, HA6, HA7, and HA9 were used for the coating of ELISA plate for the diagnosis of AIV infection. We found that this recombinant HA-ELISA could differentiate the antisera of HA3, HA5, HA6, HA7, and HA9 subtypes. In addition, recombinant HA-Western blot and HA-ELISA successfully identify a field case in which chickens were infected by AIV of HA6 subtype. This study shows the first time that recombinant HA protein expressed by a prokaryotic system can be used in differentiation of subtypes of antisera of AIV. The application of this recombinant HA-ELISA assay might help to improve the conventional diagnosis method of AIV, and to provide a rapid and specific tool for the subtype-differentiation of AIV.
中文摘要.......................................................I
英文摘要......................................................II
目次.........................................................III
表次...........................................................V
圖次..........................................................VI
第一章 緒言...................................................1
第二章 文獻探討...............................................3
2-1歷史背景....................................................3
2-2目前之流行狀況與疫情........................................4
2-3病毒特徵....................................................7
2-4結構、形態與功能............................................8
2-5複製過程...................................................12
2-6抗原特性...................................................12
2-7宿主範圍...................................................14
2-8診斷方法...................................................17
2-9 酵素連結免疫吸附分析 (ELISA) 之應用原理...................23
第三章 材料與方法............................................25
3-1 高免血清之製備............................................25
3-1.1 病毒來源................................................25
3-1.2 病毒增殖................................................25
3-1.3 偵測抗原力價............................................26
3-1.4 油質乳劑不活化疫苗之製備................................26
3-1.5 偵測抗體力價............................................27
3-1.6 疫苗接種................................................28
3-1.7 十五種HA血清亞型之抗原與抗體交叉反應試驗................28
3-2 重組表現載體之構築........................................28
3-2.1 增幅基因................................................28
3-2.2 重組基因之選殖..........................................30
3-2.3 重組質體之表現與確認....................................33
3-2.4 重組蛋白之應用..........................................36
3-2.5 應用重組蛋白偵測野外血清................................38
第四章 結果..................................................39
4-1高免血清之置備.............................................39
4-1.1 高免血清之抗體力價......................................39
4-1.2 15種HA血清亞型之抗原與抗體交叉反應試驗.................39
4-2 重組表現載體之構築........................................39
4-2.1 HA基因之選殖與表現載體之構築...........................39
4-2.2 重組蛋白之表現與純化....................................40
4-2.3 重組蛋白之特異性分析與確認..............................41
4-2.4 ELISA plate之架構與分析................................41
4-2.5 應用重組蛋白偵測野外血清................................42
第五章 討論..................................................63
參考文獻......................................................69
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1. 應用反轉錄聚合鏈反應診斷家禽流行性感冒病毒及區分血清亞型
2. 台灣地區鴨鵝小病毒之核酸序列分析與酵素連結免疫吸附分析(ELISA)套組之開發研究
3. 應用間接酵素結合免疫吸附法(ELISA)測定家禽流行性感冒血清抗體
4. 建立以競爭性酵素連結免疫吸附分析法快速診斷H5亞型高病原性家禽流行性感冒之技術
5. H5亞型高病原性家禽流行性感冒之單源抗體特性分析及其在以阻斷性ELISA快速診斷H5亞型血清技術之應用
6. 以桿狀病毒表現系統表現禽流感病毒之NA蛋白,並應用於NA亞型抗體之快速區分診斷
7. 家禽流行性感冒病毒核蛋白之核酸序列分析與酵素連結免疫吸附分析(ELISA)套組之開發
8. 以合成胜肽ELISA及阻斷性ELISA快速診斷家禽流行性感冒病毒H5及H7亞型抗體
9. 家禽流行性感冒病毒H5亞型ELISA診斷試劑之開發及垂直傳播試驗
10. 開發豬萎縮性鼻炎酵素連結免疫吸附法與不同佐劑應用於豬萎縮性鼻炎次單位疫苗的效力評估
11. 大腸桿菌表現雞傳染性喉頭氣管炎病毒醣蛋白在酵素連結免疫吸附分析之應用
12. 檢測抗家禽流行性感冒病毒NS1蛋白之抗體以區分感染野外毒與被免疫之鷄隻
13. 製備抗家禽流行性感冒病毒H6亞型血球凝集素之單源抗體
14. 以桿狀病毒表現系統表現禽流感病毒之NA蛋白,並應用於NA亞型抗體之快速區分診斷
15. 家禽流行性感冒病毒特性與傳播途徑以及食品衛生措施之研擬
 
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