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研究生: 汪宗明
Zong-Ming Wang
論文名稱: 雞蛋IgY抗體的生產與純化研究
Production and purification of IgY polyclonal antibodies in egg yolk of chicken
指導教授: 王玉麒
Wang, Yu-Chie
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2012
畢業學年度: 101
語文別: 中文
論文頁數: 166
中文關鍵詞: IgY雞蛋蛋黃抗體免疫球蛋白抗原吸附管柱
英文關鍵詞: IgY, egg yolk, antibody, immunoglobulin, antigen, antigen-adsorption column
論文種類: 學術論文
相關次數: 點閱:1040下載:25
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  • IgY是雞蛋蛋黃中的主要抗體,相較於以哺乳類動物生產IgG多株抗體,以雞蛋生產IgY多株抗體不但方式更為簡便、成本較低廉、產量也較為豐富,且程序也較符合人道要求。此外,由於鳥類與哺乳類動物的親緣關係較遠,IgY在醫學應用上能減少偽陽性反應的發生,因此IgY多株抗體具有基礎研究、一般檢驗、醫學診斷與治療等多方面用途的應用潛力。
    在本研究中,我們一方面以小鼠的IgM免疫球蛋白為模式抗原,對雞隻進行免疫注射,以探討生產專一IgY抗體之適當條件;另一方面也發展從液態蛋黃及冷凍蛋黃中粗萃取IgY的方法;此外,我們也利用基因工程的方式,生產抗原的重組蛋白質,並將其製備成吸附管柱,以純化獲得純度、效價及專一性皆優良的IgY抗體樣品。
    本研究已成功建立以雞隻生產小鼠免疫球蛋白之IgY抗體的模式,可做為以雞隻生產其他相關抗原之IgY多株抗體的參考;我們也已開發出快速、經濟、有效的IgY萃取與純化方法,除了顯著提升萃取蛋黃IgY的效率與便利性之外,更能增進IgY抗體的應用範圍。

    IgY is the major antibody in the egg yolk of chicken. There are so many advantages of IgY, the yield in the yolk is abundant, the cost of maintaining hens is low, the using of non-invasive methods to isolate IgY from yolks is more humane, and the false positive result is reduced due to large phylogenetic distance between mammals and birds, so that IgY is a useful tool for basic research, inspection, medical diagnosis and treatment.
    In this study, we have injected mouse IgM into hens, in order to produce antigen-specific IgY and investigate the appropriate production conditions. On the other hand, we have developed two methods for extracting IgY from liquid form egg yolk and frozen egg yolk respectively. Furthermore, we produced a recombinant antigen protein by genetic engineering, then manufactured an antigen-adsorption column to purify IgY which is high purity, titer and specificity.
    We have successfully established a production mode of chicken IgY against mouse immunoglobulins, which could be used as a useful reference for the production of IgY antibodies directed to other relevant antigents. We have also developed rapid, economic and effective IgY extraction and purification protocols. These protocols could not only significantly improve the efficiency and the convenience of IgY extraction from egg yolk, but also extend the applicability of IgYantibodies.

    摘要 .................................................... 1 Abstract ............................................... 2 第一章 前言 .............................................. 3 一、動物免疫防禦機制 ....................................... 3 二、抗體的應用 ............................................ 4 三、鳥類的免疫系統 ......................................... 6 四、鳥類抗體的多樣性 ....................................... 7 五、鳥類的抗體 ............................................ 8 (1) 鳥類的抗體介紹 ........................................ 8 (2) IgY結構與特性 ........................................ 9 (3) 雞蛋蛋黃中的IgY ...................................... 11 (4) 其他形態的IgY ....................................... 11 六、蛋黃IgY的應用 ........................................ 12 七、IgY萃取方法 .......................................... 13 八、研究目的 ............................................. 15 第二章 快速、有效與低成本的蛋黃IgY萃取方法 .................... 20 第一節 摘要 ............................................. 20 第二節 緒言 ............................................. 22 第三節 材料與方法 ......................................... 25 1. 蛋黃的收集 ........................................... 25 2. 蛋黃IgY的粗萃取 ....................................... 25 3. 蛋黃IgY的硫酸銨鹽析沉澱 ................................ 25 4. SDS膠體電泳分析 ....................................... 26 5. 蛋黃IgY的定量分析 ..................................... 26 6. 蛋白質定量 …………………………………………… 27 7. 統計分析 ……………………………………………… 28 第四節 結果 ……………………………………………………. 29 1. 蛋黃萃取溶液的測試 ……………………………… 29 2. 粗萃取蛋黃IgY的攪拌時間測試 …………………… 32 3. pH值對蛋黃IgY的萃取效果探討 …………...…… 35 4. 蛋黃稀釋液於攪拌期間的pH值變化情形 ……… 40 5. 蛋黃粗萃液的硫酸銨鹽析沉澱測試 ………………… 42 第五節 討論 …………………………………………………… 46 第三章 冷凍蛋黃IgY的簡易、有效萃取方法 ……..…… 54 第一節 摘要 …………………………………………………… 54 第二節 緒言 …………………………………………………… 56 第三節 材料與方法 ……………………………………… 59 1. 蛋黃的收集 ………………………………………… 59 2. 冷凍蛋黃的氯仿萃取 …….………………………… 59 3. 蛋黃IgY的硫酸銨鹽析沉澱 ……………………… 59 4. SDS膠體電泳分析 ……………………………….… 59 5. 蛋黃IgY的定量分析 …………………………….… 60 6. 蛋白質定量 …………………………..…………….… 60 7. 統計分析 …………………………………………… 60 第四節 結果 …………………………………………………… 61 1. 冷凍蛋黃萃取IgY的過程 ……………………….… 61 2. 氯仿使用量與蛋黃粗萃取液的蛋白質分析 ……… 63 3. 蛋黃粗萃液的硫酸銨鹽析沉澱測試 ……………… 66 第五節 討論 …………………………………………………… 69 第四章 由雞隻蛋黃生產小鼠免疫球蛋白之多株抗體 ………… 74 第一節 摘要 …………………………………………………… 74 第二節 緒言 …………………………………………………… 75 第三節 材料與方法 ……………………………………… 78 1. 雞隻免疫注射 …………………………………… 78 2. 雞隻靜脈抽血 …………………………………… 78 3. 蛋黃的收集 ……………………………………… 79 4. 蛋黃IgY的氯仿萃取 …………………….………… 79 5. 血清與蛋黃中的專一性IgY效價檢測 …………… 79 6. SDS膠體電泳分析 ………………………………… 80 7. 免疫轉漬實驗 ………………………………………… 81 第四節 結果 …………………………………………………… 82 1. 專一性IgY在雞隻血清與蛋黃中的表現情形 ……… 82 2. 蛋黃中IgY的專一性測試 …………………………… 87 第五節 討論 …………………………………………………… 89 第五章 抗原吸附層析純化蛋黃中IgY ………………………… 97 第一節 摘要 ……………………………………………..…….. 97 第二節 緒言 ………………………………………………. 98 第三節 材料與方法 ………………………………………….. 100 1. 萃取Balb/c小鼠B細胞mRNA .…………………… 100 2. 聚合酶連鎖反應 ……………………………………… 101 3. DNA電泳與純化目標DNA片段 ………………….. 101 4. 萃取質體pET-24a 之DNA ………………………… 102 5. 限制酵素反應 ………………………………………… 102 6. 選殖載體構築 ………………………………………… 103 7. 製備大腸桿菌勝任細胞(competent cell)………………. 103 8. 將選殖載體轉形至勝任細胞 ………………………… 104 9. 大腸桿菌表現菌株製備 ……………………………… 104 10. 將選殖載體轉形至表現菌株 ……………………… 105 11. 誘導表現重組蛋白質 ………………………………. 106 12. IPTG濃度對誘導表現效果測試 …………………… 107 13. 大規模誘導重組蛋白質並確認細菌內的存在形式 … 107 14. 萃取重組蛋白質 …………………………………. 108 15. 重組蛋白質定量分析 ……………………………… 109 16. 製作抗原吸附管柱 ……………………………… 109 17. 雞隻免疫注射 ……………………………………… 110 18. 蛋黃的收集與萃取蛋黃中IgY …………………….. 110 19. 純化專一性IgY …………………………………….. 110 20. 純化後IgY樣品對不同抗原的結合能力測試 ….. 111 21. IgY純化前後的抗體效價比較 …………………… 112 22. SDS膠體電泳分析 ……………………………….… 113 23. 免疫轉漬實驗 …………………………………….... 113 第四節 結果 …………………………………………………… 114 1. 表現載體的構築與大腸桿菌轉殖 ………………..… 114 2. 以IPTG誘導細菌表現rIgM-HC的情形 ………… 120 3. 抗原吸附管柱製作 ……………………………… 122 4. 純化專一性IgY ……………………………………… 123 5. 分析純化後的專一性IgY …………………………… 125 第五節 討論 …………………………………………………… 132 第六章 總結 ………………………………………………….… 140 參考文獻 ………………………………………………..……… 142 附錄 ……………………………………………………………… 154 附錄一、縮寫對照表 …………………………………..…… 154 附錄二、中英對照表 ………………………………………… 156 附錄三、膠體電泳分析之溶液配方 ………………………..… 161 附錄四、免液化學實驗之試劑配方 ………………………… 163 附錄五、微生物培養基配方 ………………………………… 165 附錄六、rIgM-HC的核甘酸序列 ………………………… 166 圖目錄 第一章 前言 圖一、IgG、IgY、IgY(△Fc)的結構比較圖。…………… 17 圖二、基因轉換的可能機制。…………………………………. 18 圖三、基因轉換過程示意圖。…………………………………. 19 第二章 快速、有效與低成本的蛋黃IgY萃取方法 圖一、兩種蛋黃粗萃液的蛋白質電泳分析結果。………… 31 圖二、不同攪拌時間的IgY萃取效果比較。……………… 34 圖三、不同pH條件的蛋黃粗萃液之外觀情形。….…… 37 圖四、將不同pH條件之蛋黃粗萃液進行電泳分析的結果。... 39 圖五、蛋黃稀釋液於攪拌期間的pH值變化情形。...….. 41 圖六、不同鹽析產物的電泳分析結果。…………………….… 45 第三章 冷凍蛋黃IgY的簡易、有效萃取方法 圖一、冷凍蛋黃的粗萃取過程示意圖。……………………… 62 圖二、氯仿處理之蛋黃粗萃取液的電泳分析結果。………… 65 圖三、蛋黃IgY經鹽析沉澱後的電泳分析結果。…………… 68 圖四、冷凍蛋黃的IgY萃取流程圖。…………………………. 73 第四章 由雞隻蛋黃生產小鼠免疫球蛋白之多株抗體 圖一、雞隻血清中專一性IgY的效價表現情形。……….… 84 圖二、雞蛋蛋黃中專一性IgY的效價表現情形。…………… 85 圖三、蛋黃與血清中專一性IgY的趨勢比較。……………… 86 圖四、蛋黃中IgY針對不同免疫球蛋白的免疫轉漬分析。… 88 第五章 抗原吸附管柱純化蛋黃中IgY 圖一、rIgM-HC示意圖、DNA電泳分析與表現載體圖譜。… 115 圖二、rIgM-HC的胺基酸序列。……………………………. 117 圖三、rIgM-HC在三種大腸桿菌菌株表現情形。………… 118 圖四、rIgM-HC在菌體中表現的存在形式。……………… 119 圖五、IPTG濃度對rIgM-HC表現的影響。………………… 121 圖六、專一性IgY的純化情形。…………………………..… 124 圖七、IgY樣品純化前後的蛋白質分布情形。……………… 127 圖八、純化後的IgY樣品對各型免疫球蛋白的免疫轉漬分析。……………………………………………..… 128 圖九、純化後IgY樣品針對各型免疫球蛋白的辨識情形。… 130 圖十、純化前後IgY效價表現差異情形。………………… 131 圖十一、rIgM-HC與各型小鼠免疫球蛋白重鏈部分的胺基酸序列比對。…………………………………………….. 139 表目錄 第二章 快速、有效與低成本的蛋黃IgY萃取方法 表一、兩種蛋黃粗萃取液的比較。……………………….… 30 表二、低溫攪拌時間對蛋黃IgY及蛋白質的萃取效果比較。 33 表三、不同pH條件下,蛋黃粗萃液的IgY與蛋白質含量比較。…………………………………………………. 38 表四、蛋黃粗萃液的鹽析沉澱結果。……………………….... 44 表五、不同IgY萃取方法的比較。…………………………….. 53 第三章 冷凍蛋黃IgY的簡易、有效萃取方法 表一、不同氯仿使用量對蛋黃成分萃取效果的比較。…… 64 表二、蛋黃IgY的鹽析沉澱結果。…………………..…… 67 第四章 由雞隻蛋黃生產小鼠免疫球蛋白之多株抗體 表一、各種生產雞隻專一性IgY的注射過程。…………… 95 表二、小鼠IgM重鏈與小鼠、兔、羊之IgG1重鏈之胺基酸序列的BLAST結果。……………………………….. 96 第五章 抗原吸附管柱純化蛋黃中IgY 表一、製備rIgM-HC吸附管柱的情形。………………….. 122 表二、各種小鼠免疫球蛋白的重鏈與輕鏈胺基酸序列的BLAST結果。…………………………………...... 137

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