臺灣博碩士論文加值系統

腸病毒71型 ( Enterovirus-71, EV-71)主要感染人類嬰幼兒，重症者常會出現神經症狀甚至死亡。如何有效地預防 EV-71 感染是目前人類流行病學最重要的課題之一。本實驗材料病毒取自高雄長庚紀念醫院重症兒童病患所分離到的腸病毒71型病毒，經稀釋為 8×108 pfu後作為抗原，分別以弗氏佐劑( Freund's adjuvant ) 、油質佐劑 ( oil adjuvant ) 及鋁膠鹽佐劑 ( Al-gel adjuvant ) 等三種不同型式的佐劑配合不活化腸病毒71型疫苗進行免疫，建立來亨產蛋雞生產抗腸病毒71型的專一性蛋黃免疫球蛋白 ( egg yolk immunoglobulin ; IgY ) 之生產模式；並以弗氏佐劑 ( Freund's adjuvant )配合不活化腸病毒71型進行屏科大青殼蛋雞一號免疫，憑估以屏科大青殼蛋雞一號生產蛋黃免疫球蛋白 ( IgY ) 之可行性。使用弗氏佐劑之試驗結果為血清中抗體的高峰期約為免疫後第8至10週，抗體最高值為7.3；而蛋黃中抗體的高峰期約為免疫後第8至11週，抗體最高值為7.1。使用油質佐劑配合病毒抗原之試驗結果為血清中抗體的高峰期約為免疫後第8至10週，抗體最高值為8.2；而蛋黃中抗體的高峰期約為免疫後第9至11週，抗體最高值為8.10。使用鋁膠鹽佐劑配合病毒抗原之試驗結果為血清中抗體的高峰期約為免疫後第8至10週，抗體最高值為8.1；而蛋黃中抗體的高峰期約為免疫後第9至11週，抗體最高值為7.9。屏科大青殼蛋雞

一號使用弗氏佐劑配合病毒抗原之試驗結果為：血清中抗體的高峰期約為免疫後第7至11週，抗體最高值為7.4；而蛋黃中抗體的高峰期約為免疫後第8至11週，抗體最高值為7.2。實驗最後選殖腸病毒71型VP1分子，未來可再進一步分析其親源關係及表現蛋白質。本實驗結果可提供未來被動免疫抗EV-71 IgY商品化生產模式之參考。

Enterovirus-71 ( EV-71 ), the newest member of picornavirudae, is notable for its etiological role in epidemics of severe neurological disease in children. The virus has been widespread in 1998 and caused the death of 78 children in Taiwan. It will cause the syndromes of diarrhea, foot and mouth disease, aseptic meningitis and poliomyelitis in the infection of children below 5 years old. Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulin ( IgY ) from immunized hens have so far received scant attention in research. The IgY have been evaluated in the reagents of diagnostics and therapy of gastrointestinal diseases in recent years. It’s have been proved to be more economic, easier in purification steps, higher yields, and non-invasive to animals. This study aims at detect the best anti-EV-71 IgY producing stage. We use cell supernatant inactivated EV-71 containing as antigen to inoculate the experimental animals including Leghorn hens and Green Eggshell chicken, and the titers of anti-EV71 IgY in the serum and yolk were detected by neutralization test.
In experimental of Leghorn hen groups were immunized three times by inactivated EV-71 with three different kinds of adjuvant ( Freund’s adjuvant, oil adjuvant and Al-gel adjuvant ), and then the eggs and blood samples were collected every weeks. The results revealed that all of trail groups titers of IgY against EV-71 in serum and egg yolk were increased from the second week after the first immunization, and reached the highest title at the 8-10th week in serum, each of different kinds of adjuvant ( Freund’s adjuvant, oil adjuvant and Al-gel adjuvant ) trail groups reach the highest titers of IgY in serum were 7.3, 8.2 and 8.1, respectively. Moreover, each of trail groups reached the highest titers of IgY in egg yolk were at the 8-11, 9-11 and 9-11 weeks, respectively, and the highest IgY titers of NT in egg yolk were 7.1, 8.1 and 7.9, respectively. Furthermore, in experimental of Green Eggshell chicken group, chicken were immunized four times by inactivated EV-71 with Freund’s adjuvant. The eggs and blood samples were collected every week. The titers of IgY against EV-71 in serum and egg yolk were increased from the second week after first immunization, and reached the highest immunity response at the 7-11 and 8-11 weeks. The highest IgY titers of NT in serum and egg yolk were 7.4 and 7.2 respectively. The data obtained from this study will construct a model for producing high titer of IgY against EV71 by Leghorn hens or Green Eggshell chicken.

中文摘要 Ⅰ
Abstract Ⅲ
誌謝 Ⅴ
目錄 Ⅵ
圖表目錄 Ⅸ
第1章 前言 1
第2章 文獻回顧 3
2.1腸病毒71型 3
2.1.1 腸病毒71型的簡介 3
2.1.2 腸病毒71型病毒學分類 3
2.1.3 腸病毒71型病毒結構及特性 4
2.1.4 腸病毒71型的致病機制 5
2.1.5 臨床症狀 5
2.1.6 流行病學 6
2.1.7 腸病毒71型感染的診斷 7
2.1.8 腸病毒之預防及治療 7
2.2蛋黃免疫球蛋白 8
2.2.1 蛋黃免疫球蛋白的命名 8
2.2.2 蛋黃免疫球蛋白的優點 8
2.2.3 蛋黃免疫球蛋白 ( IgY ) 分子的構造 9
2.2.4 蛋黃免疫球蛋白 ( IgY ) 之來源 10
2.2.5 IgY的分子安定性 11
2.2.6 分離純化IgY 之方法 13
2.2.7 IgY 之應用 14
2.3現今疫苗發展概況 16
第3章 材料與方法 18
3.1 試驗材料 18
3.2 試驗方法 18
3.2.1 細胞培養 18
3.2.2 冷凍細胞 18
3.2.3 解凍細胞 19
3.2.4 病毒培養 19
3.3腸病毒71型之確認 19
3.3.1 腸病毒71型RNA之萃取 19
3.3.2 確認腸病毒71型病毒之引子對設計 20
3.3.3 聚合酵素連鎖反應 20
3.3.4 核酸定序與比對分析 20
3.4病毒之毒力測定－TCID50 21
3.5產蛋來亨雞及屏科大青殼蛋雞一號免疫試驗 21
3.5.1 抗原之製備 21
3.5.2 以不同佐劑配合腸病毒71型抗原進行免疫試驗 21
3.5.3 血清待測樣本的分離與保存 22
3.5.4 免疫雞隻血清之血清中和試驗 22
3.5.5 蛋黃待測樣本的分離與保存 22
3.5.6 免疫雞隻蛋黃之中和試驗 22
3.5.7 實驗結果統計分析 23
3.6腸病毒71型VP1分子之選殖 23
3.6.1 腸病毒71型RNA之萃取 23
3.6.2 腸病毒71型VP1片段引子對設計 23
3.6.3 聚合酵素連鎖反應 24
3.6.4 PCR產物之純化 24
3.6.5 連結（ligation）及轉型（transformation） 24
3.6.6 篩選（screening） 25
3.6.7 DNA定序與比對分析 25
第4章 結果 26
第5章 討論 47
參考文獻 57
附錄一 Immunoglobulin Y 72
附錄二 實驗用藥品及套組 73
附錄三 QIA quick® gel extraction kit 操作手續 75
附錄四 QIAprep® Spin Minprep Kit操作手續 76
附錄五 pGEN T-easy Vector map 77
作者簡介 78

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