腸病毒71型為季節性的傳染病，常引起手口足症，對於五歲以下的幼童更具有高度的傳染力。我們已可利用桿狀病毒系統生產出腸病毒71型的類病毒顆粒(virus-like particle, VLP)，這些VLP能引發小鼠良好的抗體反應，且產生的抗體對不同基因型的腸病毒71型皆具有中和能力，顯示VLP有潛力作為腸病毒71型的疫苗候選者。本研究中，我們探討腸病毒VLP最佳生產條件，包含病毒株與細胞株與培養基組合，最佳感染細胞濃度及溶氧量等，期望能增加腸病毒VLP的單位體積產率。

目錄
摘要 I
目錄 V
圖目錄 VIII
1-1腸病毒(Enterovirus) 71型 9
1-1-1 腸病毒流行病學 9
1-1-2 腸病毒71型 10
1-2 桿狀病毒/昆蟲細胞表現系統 11
1-2-1 昆蟲桿狀病毒(baculovirus)簡介 11
1-2-2 昆蟲細胞 12
1-2-3桿狀病毒表現載體的發展與應用 13
1-2-4 腸病毒71型疫苗發展 14
1-3 類病毒顆粒(virus-like particles，VLP) 15
1-4 研究動機 16
2-1 細胞培養 23
2-1-1昆蟲細胞培養 23
2-1-2哺乳動物細胞培養 24
2-2 重組桿狀病毒 24
2-2-1 重組桿狀病毒載體 24
2-2-2 重組桿狀病毒放大 24
2-2-3 終點稀釋法定量病毒效價 25
2-2-4 腸病毒71型類病毒顆粒的生產 25
2-2-5 生物反應器生產腸病毒71型類病毒顆粒 25
2-3純化胞內腸病毒71型類病毒顆粒 25
2-3-1 胞內腸病毒71型類病毒顆粒的萃取 26
2-3-2 氯化銫(CsCl)不連續密度梯度離心 26
2-3-3 氯化銫(CsCl)等密度梯度離心 27
2-3-4 以蛋白質定量法定量類病毒顆粒的總蛋白濃度 27
2-4純化胞外腸病毒71型類病毒顆粒 27
2-4-1 胞外腸病毒71型類病毒顆粒的收集 28
2-4-2 切向流過濾法濃縮及純化腸病毒類病毒顆粒 28
2-4-3 氯化銫(CsCl)不連續密度梯度離心 28
2-5 腸病毒 71 型 28
2-5-2 腸病毒的純化 29
2-6 VLP特性分析方法 30
2-6-1 SDS-PAGE 30
2-6-2 西方點墨法(western blotting) 31
2-6-3酵素連結免疫吸附分析法(Enzyme-Linked Immunosorbent Assay ) 32
2-6-5 穿透式電子顯微鏡(Transmission Electron Microscopy，TEM) 33
2-6-6 免疫金染色及穿透式電子顯微鏡觀察 33
2-7小鼠免疫實驗分析 34
2-7-1小鼠免疫及血清樣本收集 34
2-7-2 抗體效價 34
2-7-3 中和效價 35
2-7-4 交叉保護效果(cross reaction)分析 36
2-8 統計分析 36
第三章 實驗結果 39
3-1類病毒顆粒最佳生產條件之探討 39
3-1-1 病毒株與細胞株組合篩選 39
3-1-2最佳感染濃度探討 40
3-1-3反應器生產類病毒顆粒 41
3-2類病毒顆粒定性分析 41
3-2-1 氯化銫不連續梯度離心 42
3-2-2 氯化銫連續梯度離心 42
3-2-3蛋白質鑑定身分比對 43
3-2-4穿透式電子顯微鏡觀察 43
3-3類病毒顆粒做為疫苗的初步免疫性質探討 44
3-3-1小鼠免疫及血清樣品採集 45
3-3-2抗體效價分析 45
3-3-3血清中和效價(neutralization titer)分析 46
3-3-4抗體對於不同腸病毒株的交叉保護 46
第四章 結論 57
第五章 未來展望 58
5-1 生產程序的改善 58
5-2純化程序的改善 58
5-3類病毒顆粒保存環境的最佳化及穩定性之分析 59
第六章 參考文獻 60

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