小兒麻痺病毒(Poliovirus)的感染會引起嬰兒與幼童發燒、頭痛，甚至侵入中樞神經系統造成永久性的殘疾。因小兒麻痺症無特效藥物可醫治，最佳的防疫方法就是接種疫苗。目前市面上最常見的疫苗為沙克疫苗與沙賓口服疫苗，雖然免疫效果十分良好但這兩種疫苗為真實病毒經由突變或是化學處理後作為抗原施打，皆有疫苗中病毒毒性恢復的危險。自1988年世界衛生組織以疫苗策略推行小兒麻痺根除計畫以來，已獲得全球性的防疫成果，但在根除計畫的尾聲，減少疫苗所帶來的隱憂是十分重要的環節。類病毒顆粒疫苗並不含有病毒的遺傳物質，可使用基因工程技術進行生產，作為疫苗無毒性復發與病毒突變等問題，因此欲完全根除小兒麻痺病毒，以類病毒顆粒作為疫苗是相當有利的工具。
本實驗室先前已成功的利用昆蟲細胞/重組桿狀病毒表現系統生產腸病毒71型類病毒顆粒疫苗，其品質與產量相當穩定也可引起良好的抗病毒免疫效果。在本研究中我們希望根據先前的經驗，以昆蟲細胞/重組桿狀病毒系統進行同為腸病毒屬的小兒麻痺類病毒顆粒疫苗開發。我們以最易衍生疫苗病毒感染的第二型小兒麻痺病毒為優先，利用舊有的重組桿狀病毒載體為基礎，分別以polyhedrin啟動子驅動小兒麻痺病毒P1外殼蛋白與CMV、ETL或vp39啟動子驅動3CD水解酶，藉由flashBACULTRATM系統製作成重組桿狀病毒。我們從這些建構中篩選出類病毒顆粒產量與質量最好的BacU-PV2-P1-vp39-3CD病毒，並將此病毒建構應用於第一型與第三型小兒麻痺類病毒顆粒的生產。我們證實以vp39啟動子驅動3CD水解酶的病毒建構在感染High FiveTM細胞後，第二型與第三型病毒的P1外殼蛋白可以被正確的切割成VP0、VP3以及VP1三個結構蛋白，純化後可以在穿透式電子顯微鏡下觀察到大小約30 nm、空心的二十面體顆粒；然而第一型類病毒顆粒的產量與質量卻不如預期。
因此後續實驗我們將mRNA表現量較高的第三型3CD水解酶置換到第一型小兒麻痺類病毒顆粒的重組載體上，由初步生產結果可發現，此重組載體可生產出切割較為完整的類病毒顆粒。未來的實驗我們將第一型外殼蛋白搭配第三型3CD水解酶的重組桿狀病毒感染High FiveTM細胞放大生產，以蔗糖連續梯度超高速離心純化，期待能得到組裝完整的標準品，加速根除小兒麻痺病毒的實現。

Poliovirus (PV), which may elicit the fever or headache and leads to irreversible paralysis of children less than 5 years of age, is a highly infectious disease caused by a virus. However, there is no cure for Poliomyelitis, the only efficient way to protect children from being transmitted is vaccination. There are two vaccines that have been commonly used recently: Salk inacticated polio vaccine (IPV) and Sabin oral polio vaccine (OPV), while both IPV and OPV possess some criticial disadvantages. The immune response induced by IPV is not comprehensive (especially in the instestines) that may cause indirect infection through faeces. Though OPV is effective and inexpensive, in rare cases the live attenuated vaccine-virus in OPV can cause paralysis, which is called vaccine-derived poliovirus (VDPV). From 1988, World Health Organization (WHO) launched the Global Polio Eradication Initiative (GPEI) for the purpose of eliminating wild-type poliovirus in the world, has already helped countries to make huge progress in protecting the global population from this debilitating disease. Virus-like particle (VLP), which contains no viral genetic material, is non-infectious and can ignore the risk of virus mutation. Therefore, to remove poliovirus completely, VLPs are forceful and powerful tools.
Poliovirus has an RNA genome that can be translated into a polyprotein, and the viral P1 polyprotein is cleaved by 3CD protease into individual structural proteins (VP1, VP3 and VP0). By mimicking the natural process, we developed virus-like particle (VLP) vaccines by the baculovirus/insect cells expression system combined with flashBACULTRATM system to co-express P1 and 3CD derived from PV-type 1, type2 and type 3. First of all, we select the promoter to drive 3CD protease among CMV, ETL, and vp39, and the recombinant baculovirus BacU-PV2-P1-vp39-3CD express VLPs with most efficient P1 cleavage. After production and purification of VLPs by continuous sucrose ultracentrifugation, we can clearly observe icosahedron with diameter 30 nm, empty particles under the transmission electron microscope (TEM). However, we cannot obtain VLPs of poliovirus type 1 after purification.
For the following experiment, we replace 3CD protease gene of type 1 on recombinant baculovirus construction with 3CD protease gene of type 3, which has the better expression. With this construction, the cleavage of P1 polyprotein of type 1 improves and we hope to purify successfully to accelerate the eradication of poliovirus in the foreseeable future.

摘要 I
Abstract II
目錄 III
圖表目錄 VI
第一章 文獻回顧 1
1-1 小兒麻痺病毒 1
1-1-1 小兒麻痺病毒簡介 1
1-1-2 小兒麻痺症的流行病學 2
1-2 小兒麻痺疫苗發展 3
1-2-1 沙克疫苗 3
1-2-2 沙賓疫苗 4
1-2-3 小兒麻痺病毒根除計畫 5
1-2-4 目前小兒麻痺疫苗所面臨的問題 5
1-2-5 類病毒顆粒疫苗 7
1-3 昆蟲細胞/重組桿狀病毒表現系統 9
1-3-1 昆蟲細胞/重組桿狀病毒表現系統的發展 9
1-3-2 昆蟲細胞/重組桿狀病毒表現系統的應用 10
1-3-3 flashBACTM桿狀病毒表現系統 12
1-3-4 flashBACULTRATM桿狀病毒表現系統 12
1-4 Gibson Assembly 13
1-5 研究目的 14
第二章 實驗材料及方法 23
2-1 昆蟲細胞培養 23
2-2 重組桿狀病毒 23
2-2-1 重組載體的系統與建構 23
2-2-1-1 第二型小兒麻痺類病毒顆粒載體的建構 24
2-2-1-2 第一型與第三型小兒麻痺類病毒顆粒載體的建構 24
2-2-2 重組桿狀病毒的建構 25
2-2-3 重組桿狀病毒的放大 25
2-2-4 重組桿狀病毒感染效價 26
2-3 小兒麻痺類病毒顆粒 26
2-3-1 小兒麻痺類病毒顆粒生產 26
2-3-2 小兒麻痺類病毒顆粒純化流程 27
2-3-2-1 切向流過濾(Tangential Flow Filtration) 27
2-3-2-2 蔗糖連續濃度梯度超高速離心純化(Continuous sucrose ultracentrifugation) 28
2-4 實驗分析方法 28
2-4-1 SDS-PAGE凝膠電泳 28
2-4-2 西方點墨法(Western Blot) 29
2-4-3 Bradford 蛋白質濃度測定 30
2-4-4 免疫酵素連結吸附分析法(ELISA) 30
2-4-5 穿透式電子顯微鏡 31
2-5 RT-qPCR絕對定量分析mRNA表現 32
2-6 終點稀釋法(End-point dilution method)篩選單株病毒 33
2-7 以Gibson Assembly進行小兒麻痺第一型類病毒顆粒的載體建構 34
2-7-1 Gibson Assembly之載體(vector)的建構 34
2-7-2 Gibson Assembly Reaction 34
2-7-3 Gibson Assembly重組載體的轉染與放大 37
第三章 實驗結果與討論 42
3-1 重組桿狀病毒的建構與啟動子的篩選 42
3-2 小兒麻痺類病毒顆粒的生產 43
3-3 小兒麻痺類病毒顆粒的純化 44
3-4 小兒麻痺類病毒顆粒純化樣品分析 45
3-4-1 以凝膠電泳分析純化標準品 45
3-4-2 以免疫酵素連結吸附法偵測類病毒顆粒產量 46
3-4-3 以穿透式電子顯微鏡觀察類病毒顆粒 46
3-5 第一型小兒麻痺類病毒顆粒之產量優化 47
3-5-1 更改第一型小兒麻痺類病毒顆粒的生產條件 47
3-5-2 以終點稀釋法進行第一型單株桿狀病毒的篩選 47
3-6 外殼蛋白P1與水解酶3CD 基因表現的定量分析 48
3-7 重新設計第一型重組桿狀病毒之建構 49
3-7-1 使用Gibson Assebly搭配終點稀釋法進行DNA shuffling 49
3-7-2 多種基因組合之單株病毒的放大與單株病毒篩選 50
3-7-3 建構以不同型別3CD水解酶切割PV1外殼蛋白之重組病毒 51
3-8 第一型小兒麻痺類病毒顆粒的生產 51
3-9 結論 52
第四章 未來展望 72
4-1 第一型小兒麻痺類病毒顆粒的純化 72
4-2 第二型小兒麻痺類病毒顆粒產量之提升 72
4-3 腸病毒71型與克沙奇病毒A16型類病毒顆粒產量之提升 73
第五章 參考文獻 74

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