克沙奇病毒(coxsackievirus)盛行於東亞地區，屬於腸病毒不同的血清型，且為造成手口足症的主要病原體之一，感染幼童會造成嚴重神經性併發症甚至死亡，臺灣自1998年以來已爆發三次大規模腸病毒流行，皆有數百名重症案例，近年來，也有傳出因感染腸病毒重症而死亡的案例，因此克沙奇病毒的疫苗開發更顯迫切。過去實驗室使用重組桿狀病毒/昆蟲細胞表現系統生產克沙奇A16型類病毒顆粒疫苗，其外觀結構與真實病毒相似，然而在動物實驗中卻無法引發良好的抗病毒免疫反應，推測原因為所使用的建構病毒型別上的因素，以及純化製程上的問題。因此，本研究中我們建構新型別CVA16-Shzh 05的重組桿狀病毒來提升克沙奇A16型類病毒顆粒的品質，並以管柱層析純化方式純化所生產出來的類病毒顆粒，作為疫苗抗原注射小鼠。雖然在後續小鼠免疫實驗上有引發良好的血清IgG抗體效價，然而最後的中和抗體效價實驗，卻發現這些抗體不具有保護能力。為了進一步確認其具有中和抗體效價，我們期望以不同的克沙奇A16型類病毒顆粒的純化程序，來驗證其類病毒顆粒的蛋白組成和免疫性。後續實驗的規劃上，我們將利用不同純化方式取得類病毒顆粒作為疫苗抗原，探討不同純化程序取得的疫苗是否能引發抑制克沙奇A16型病毒感染的中和抗體，以應用在未來開發雙價疫苗的策略上。

Coxsackievirus A16 (CVA16) is one of the causative pathogens of hand, foot and mouth disease (HFMD), which leads to a major public health issue across the Asia-Pacific region. It mostly affects children and causes severe neurological complications and even death. Since 1998, there have outbreak of enterovirus infection in Taiwan, resulting in hundreds of cases with severe complications and entailing the need to develop vaccines against CVA16. Using the recombinant baculovirus/insect cell expression vector system, we have developed a CVA16 vaccine candidate based on virus-like particle (VLP) that resembles the authentic virus in size, shape and composition, but failed to elicit anti-viral immune response in animal model. In this study we used new viral strain of CVA16 to construct baculovirus and developed purification process in order to improve the VLP quality and efficacy. We next evaluated the immune responses elicited by the purified CVA16 VLP. With the improved purification process, we produced CVA16 VLP to develop a vaccine candidate against enterovirus infection. In the future, we will improve purification process and scale-up for production, also evaluate the immunogenicity of combined VLP antigens of different enterovirus and eventually prove the broad protection of the multiple VLP vaccine against viral challenge in mouse model.

摘要 I
Abctract II
目錄 III
圖表目錄 VI
第一章 文獻回顧 1
1-1 腸病毒 1
1-1-1 腸病毒的分類與疫情 1
1-2 克沙奇病毒A16型 2
1-2-1 克沙奇病毒A16型的流行疫情 2
1-2-2 克沙奇病毒A16型的結構 3
1-2-3 克沙奇病毒A16型的疫苗開發 4
1-3 類病毒顆粒疫苗 5
1-4 重組桿狀病毒/昆蟲細胞表現系統 7
1-4-1 桿狀病毒 7
1-4-2 桿狀病毒表現系統的發展 7
1-4-3 flashBACGOLD™重組桿狀病毒表現系統 8
1-5 研究動機 10
第二章 材料與方法 18
2-1 重組桿狀病毒的建構 18
2-1-1 flashBACGOLD™轉殖載體的建構 18
2-1-2 桿狀病毒基因體重組 18
2-1-3 重組桿狀病毒的放大 19
2-1-4 建構生產克沙奇A16型類病毒顆粒的重組桿狀病毒 19
2-1-5 重組桿狀病毒感染效價 20
2-2 細胞培養 20
2-2-1 昆蟲細胞培養 20
2-2-2 哺乳類動物細胞培養 21
2-3 克沙奇病毒A16型類病毒顆粒的生產 21
2-4 克沙奇病毒A16型類病毒顆粒的純化 22
2-4-1 切向流過濾濃縮 22
2-4-2 羥基磷灰石管柱層析法 23
2-4-3 分子篩管柱層析法 24
2-4-4 蔗糖連續式梯度離心 25
2-4-5 Capto 管柱系統層析法 26
2-5 克沙奇病毒A16型類病毒顆粒的分析 27
2-5-1 克沙奇病毒A16型病毒感染效價 27
2-5-2 克沙奇病毒A16型的生產 27
2-5-3 Bradford蛋白質濃度測定 28
2-5-4 SDS-PAGE蛋白質分離電泳 28
2-5-5 SDS-PAGE蛋白質染色 29
2-5-6 西方點墨法 29
2-6 小鼠免疫試驗 31
2-6-1 疫苗配方及製備 31
2-6-2 疫苗抗原接種 31
2-7 小鼠免疫分析 32
2-7-1 酵素連結免疫斑點分析法(ELISPOT) 32
2-7-2 血清IgG抗體效價 33
2-7-3 血清IgG抗體效價結果判讀 34
2-7-4 血清中和抗體效價 34
第三章 實驗結果 41
3-1 更改病毒株型別對於克沙奇病毒A16型類病毒顆粒之影響 41
3-2 以管柱層析法純化克沙奇病毒A16型類病毒顆粒 41
3-2-1 克沙奇病毒A16型類病毒顆粒生產與純化過程 42
3-3 克沙奇病毒A16型類病毒顆粒的小鼠免疫抗原性 44
3-3-1 酵素連結免疫斑點分析(ELISPOT) 44
3-3-2 免疫小鼠血清中抗克沙奇病毒A16型IgG結合抗體效價 45
3-3-3 免疫小鼠血清中和抗體效價 45
3-4 以蔗糖梯度離心法純化克沙其病毒A16型類病毒顆粒 46
3-4-1以High Five™昆蟲細胞生產類病毒顆粒的純化過程 47
3-4-2以Sf-9昆蟲細胞生產類病毒顆粒的純化過程 48
3-5 克沙奇病毒A16型類病毒顆粒的小鼠免疫抗原性 49
3-5-1 酵素連結免疫斑點分析(ELISPOT) 49
3-5-2 免疫小鼠血清中抗克沙奇病毒A16型IgG結合抗體效價 50
3-5-3 免疫小鼠血清中和抗體效價 51
3-6 以Capto 陰離子管柱層析法純化克沙奇病毒A16型類病毒顆粒 53
3-6-1 克沙奇病毒A16型類病毒顆粒生產與純化過程 53
3-7 克沙奇病毒A16型類病毒顆粒的小鼠免疫抗原性 55
3-7-1 酵素連結免疫斑點分析(ELISPOT) 55
3-7-2 免疫小鼠血清中抗克沙奇病毒A16型IgG結合抗體效價 56
3-7-3 免疫小鼠血清中和抗體效價 56
3-8 結論 58
第四章 討論 77
4-1 更改克沙奇A16型病毒株型別對小鼠免疫結果之影響 77
4-2 更改以Capto管柱層析純化方法對小鼠免疫結果之影響 78
第五章 未來展望 79
第六章 參考文獻 80

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