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研究生:林玉芬
研究生(外文):Yi-Fan Lin
論文名稱:IgY的萃取、純化及功能性之探討
論文名稱(外文):IgY extraction, purification and function investigation
指導教授:連塗發陳立人陳立人引用關係
指導教授(外文):Tu-Fa LienLih-Ren Chen
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:動物科學系研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:82
中文關鍵詞:蛋黃抗體大腸桿菌蛋白質純化
外文關鍵詞:IgYEscherichia coliprotein purification
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IgY的萃取、純化及功能性之探討
本試驗旨在研發萃取及純化IgY之方法及探討特異性大腸桿菌IgY之功能性。試驗分為2部分: 試驗一為建立萃取與純化IgY之方法; 試驗二為不同蛋雞品系對大腸桿菌抗原之免疫反應及特異性大腸桿菌IgY之功能性探討。試驗一中初萃取之方式分別在雞蛋與鴨蛋2組中皆使用2種溶劑(方法一: isopropanol , 方法二: triton),鴨蛋組IgY經過isopropanol 與triton萃取後,分別獲得43%和13%蛋白質,雞蛋組則獲得61%和16%蛋白質。以硫酸銨鹽析沉澱蛋白質後,鴨蛋組之純度為53-57% (titer: 19.9-26.2 unit/mg protein),雞蛋組之純度為45-50% (titer: 24-29.5U)。以膠體過濾層析法純化後,鴨蛋組純度為89-90% (titer: 415-451U),雞蛋組純度為85-87% ( titer: 240-277.6U)。最後親和性層析法純化,鴨蛋組純度為91-92% (titer: 459-461U),雞蛋組純度為93-95% (titer: 312-470U)。以不同之有機溶劑萃取雞蛋及鴨蛋所得之結果相似,因此往後除了利用來航雞生產IgY抗體外也可使用鴨蛋,並可藉此提高本土菜鴨之利用性,此方法可得到高純度之IgY。試驗二使用商業用大腸桿菌四合一疫苗 (k88、k99、987P、F41)及民間農場分離之大腸桿菌經家畜防治所純化之抗原 (A farm抗原)為免疫來源,生產抗大腸桿菌之IgY抗體。來航雞四合一抗原組抗大腸桿菌IgY 平均為65.28 μg/ml ,A farm組抗大腸桿菌IgY 平均為48.31 μg/ml 。龍門有色蛋雞四合一抗原組抗大腸桿菌IgY 平均為55.78 μg/ml,A farm組抗大腸桿菌IgY 平均為14.4 μg/ml,每年每隻雞可生產達2000 mg 以上之anti E. coli IgY。抑菌試驗結果,來航雞與龍門有色蛋雞之四合一組皆較控制組有顯著抑制大腸桿菌效果。而A farm組則無顯著抑制效用(9和19週),且IgY濃度應在60 μg/ml以上才具抑菌效果。螢光抗體結果顯示,兩雞種中四組皆有螢光反應,其中以來航雞四合一組螢光反應最強,抗原抗體間結合強度較佳。綜合上述結果,以蛋生產IgY抗體是極具發展性之方法。

IgY extraction, purification and function investigation

The objective of this study was to investigate the method of IgY extraction, purification and the function of anti-E. coli IgY. This study included two experiments: Experiment 1 to investigation the methods of extraction and purification of IgY and Experiment 2 to study the immunization response to E. coli antigen in different breeds of chicken and anti-E. coli IgY function. Experiment 1, the yolk protein of laying hens and Tsaiya ducks were extracted using isopropanol or triton. Experiment results indicated that the extracted yolk protein using isopropanol or triton in ducks were 43% and 13%, while in hens were 61% and 16%. The purity of ducks IgY was 53-57%, titer was 19.9-26.2 unit/mg protein; in hens IgY purity was 45-50%, titer was 24-29.5U when salted out by ammonium sulfate. After purification by gel filtration chromatography, results showed that the IgY purity of ducks was 89-90% with titer 415-451U, while the IgY purity of hens was 85-87%, titer was 240-277.6U. With advanced purification by affinity chromatography, IgY purity of ducks was 91-92%, titer was 459-461U; IgY purity of hens was 93-95%, titer was 312-470U. This method can be used for the production of IgY antibody from native Taiwan Tsaiya ducks or laying hens. The purpose of Experiment 2 was to produce anti-E. coli IgY. The E. coli antigen was either from the commercial four-in-one vaccine or isolated from backyard farm (Tainan livestock disease control center). The anti-E. coli IgY production of Leghorn chickens in four-in-one group was 65.28μg/ml, while in the A farm group was 48.31μg/ml. The anti-E. coli IgY production of Lohmann chickens in the four-in-one group was 55.78μg/ml,while in the A farm group was 14.4μg/ml. The chicken produces more than 2000mg of anti-E.coli IgY per year (four in one group of Leghorn and Lohmann chickens). The inhibition of E. coli test from the four-in-one group of Leghorn and Lohmann chickens were significantly higher than that of control group, but no siginificant differences between of the Leghorn and the Lohmann chickens in the A farm group(9 and 19 weeks). The inhibition of E. coli concentration in the IgY should be at 60μg/ml. All the groups had fluorescent responses and the four-in-one group of the Leghorn chickens had the strongest. In conclusion, there is potential in using egg to produce IgY antibody.

目錄 頁次
前言………………………………………………………………….1
文獻探討…………………………………………………………….3
一、蛋黃抗體IgY之介紹………………………………………...3
1.蛋組成份及蛋黃抗體IgY的來源………………………………3
2.IgY和IgG之差異……………………………………………….4
(1)分子量…………………………………………………………...4
(2)穩定性…………………………………………………………...5
(3)酵素分解性質…………………………………………………...5
3.IgY作為抗體來源之優勢………………………………………..7
(1)免疫分析應用及反應之優點…………………………………….7
(2)抗體產量及生產方式之優點…………………………………….9
(3)符合動物福利法規……………………………………………….10
二、IgY的分離與純化…………………………………………….11
1.蛋黃IgY抗體之萃取方法……………………………………….11
2.蛋黃IgY抗體之純化方法……………………………………….14
三、家禽蛋黃抗體(IgY)之應用………………………………….17
1.被動免疫治療…………………………………………………...17
2.診斷與治療應用…………………………………………….......19
3.DNA-IgY之應用………………………………………………...20
四、大腸桿菌(Escherichia coli)之概述………………………….21
1.大腸桿菌之介紹………………………………………………...21
(1)結構與性質……………………………………………………...21
(2)大腸桿菌的分類………………………………………………...23
(3)感染及臨床症狀………………………………………………...24
材料與方法…………………………………………………………27
試驗一、不同的萃取與純化方法對IgY回收率之比較…………..27
一、試驗流程……………………………………………………..27
二、試驗材料與方法……………………………………………..28
(一)免疫計畫………………………………………………………..28
1.抗原製備及免疫流程…………………………………………....28
2.集蛋………………………………………………………………28
3.蛋黃粗萃取………………………………………………………28
4.IgY初步純化……………………………………………………..29
5.IgY純化法………………………………………………………..30
(1)膠體過濾層析法………………………………………………….30
(2)親和性層析法…………………………………………………….30
(二)IgY之檢測方法………………………………………………….31
1.蛋白質定量……………………………………………………….31
2.ELISA抗體力價分析……………………………………………..33
3.SDS-PAGE………………………………………………………...35
4.蛋白質密度膠片掃描器…………………………………………..38

試驗二、不同蛋雞品系對大腸桿菌抗原之免疫反應比較.......39
一、試驗流程...........................................39
二試驗材料與方法…………………………………………………...40
(一)免疫計畫………………………………………………………....40
1.抗原製備…………………………………………………………....40
2.免疫注射流程安排………………………………………………....40
3.集蛋流程…………………………………………………………....41
4.萃取抗體…………………………………………………………....41
(二)抗體分析項目……………………………………………………42
1.抗體力價分析………………………………………………………42
2.抗原濃度……………………………………………………………44
3.抑菌測試……………………………………………………………44
4.螢光顯微鏡分析(抗原與抗體結合鏡檢)………………………….45
5.統計分析……………………………………………………………45
結果與討論…………………………………………………………...46
試驗一、不同的萃取與純化方法對IgY回收效率之比較………….46
試驗二、不同蛋雞品系對大腸桿菌抗原之免疫反應比較………...55
結論…………………………………………………………………...74
參考文獻……………………………………………………………...75
英文摘要……………………………………………………………...81



圖 次
圖1.哺乳動物IgG與雞蛋IgY之結構圖……………………………….5
圖2.層析法之概況圖…………………………………………………14
圖3.大腸桿菌顯微鏡圖………………………………………………22
圖4.IgY以初步硫酸銨純化後之SDS- PAGE電泳圖………………...53
圖5.IgY經gel filtration 及affinity層析純化之SDS- PAGE電泳圖….54
圖6.來航雞之總IgY含量………………......………………………...59
圖7.龍門有色蛋雞之總IgY含量…………...………………………..60
圖8.來航雞和龍門有色蛋雞之抗大腸桿菌IgY含量………….........61
圖9.來航雞和龍門有色蛋雞之血清中IgG含量………….................62
圖10.來航雞和龍門有色蛋雞之抗大腸桿菌IgY含量…...................63
圖11.來航雞與龍門有色蛋雞生產之抗大腸桿菌IgY與A farm 之
大腸桿菌進行抑菌試驗之結果.................................................71
圖12.來航雞生產之抗大腸桿菌IgY與A farm之大腸桿菌螢光
抗體試驗之結果……………………………………………….72
圖13.龍門有色蛋雞生產之抗大腸桿菌IgY與A farm之大腸桿
菌螢光抗體試驗之結果………………………………….73

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