禽流感病毒的基因具有容易發生重組及突變的特性，不僅對全世界的家禽產業造成極大影響，也對人類的健康存在著危害，因此需要發展有效的疫苗來防止大規模流行。然而現今的疫苗存在著一些問題，如：減毒活疫苗具有安全的疑慮、去活化疫苗與次單位疫苗能誘發的免疫效果低，需搭配佐劑來提升疫苗的效力等。而本研究希望利用桿狀病毒載體作為新型的禽流感疫苗，以桿狀病毒作為疫苗的優點包括：高度安全性、製備容易與高免疫性，因此具有發展的潛力。於先前的研究已成功將禽流感病毒的主要抗原血球凝集素(hemagglutinin, HA)，同時呈現於桿狀病毒的套膜上，以及於哺乳動物細胞內表現，這樣的雙向型重組桿狀病毒能夠引發小鼠良好的免疫反應。在此研究中，我們將結合FLP/Frt長效型表現系統，使抗原能夠長期表現，並觀測延長抗原表現是否能增強免疫反應。在此之前，我們先確認FLPo/Frt系統是否能於C2C12細胞內發揮活性，於胞內形成環型episome的結構，結果證實能夠成功地形成episome且達70%的重組效率，並且FLPo重組酶對於C2C12細胞沒有顯著的毒性。接著，我們以FLPo/Frt統作為長效表現的主軸，分別建構兩種桿狀病毒：Bac-FCHW及Bac-FCsHW，並用以轉導HeLa細胞及C2C12細胞，我們發現長效組(Bac-FLPo/Bac-FCHW)及長效分泌組(Bac-FLPo/Bac-FCsHW)的表現期皆可達21天以上。從Native PAGE的結果也發現分泌至胞外的HA蛋白，其蛋白結構依然維持三聚體。在免疫實驗中，長效分泌組的小鼠能夠引起較強烈的體液性免疫反應，產生較多的抗HA的抗體及中和抗體。本研究為第一個以桿狀病毒搭配長效表現系統與分泌型抗原當做疫苗載體，希望對於未來以桿狀病毒載體作為禽流感疫苗有更多的幫助。

Avian influenza virus (AIV) poses a tremendous threat to the poultry industry and public health worldwide, thus entailing the development of effective vaccine to prevent pandemic. Current avian influenza vaccines have their respective problems. For example, inactivated virus and live attenuated virus vaccines have raised safety concerns. Subunit vaccines might induce less potent immunogenicity. Baculovirus is capable of expressing foreign genes in mammalian cells, and emerges as a safe and effective gene delivery vector. We have previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of AIV on the viral envelope and expressing HA after transducing mammalian cells. This dual form vaccine can elicit robust humoral and cellular immunity. Here we explore whether prolonged antigen expression can ameliorate the vaccine efficacy. We developed an FLPo/Frt-based baculovirus binary system consisting of 2 baculoviruses: one expressing FLPo recombinase and the other donor vector expressing the transgene cassette flanked by Frt sequences. Co-transduction of C2C12 cells the two vectors led to the FLPo-mediated excision of the transgene cassette from the baculovirus genome and formation of episomal DNA minicircle at an efficiency approaching 70%, without appreciable toxicity. We next constructed two done baculovirus vectors: Bac-FCHW expressing HA, and Bac-FCsHW expressing secreted form of trimeric HA. Co-transduction of HeLa and C2C12 cells with Bac-FLPo/Bac-FCHW led to sustained HA expression for at least 21 days. Co-transduction with Bac-FLPo/Bac-FCsHW conferred prolonged expression and secretion of trimeric HA into the medium. These baculovirus vectors for prolonged antigen expression/secretion may be novel candidates to enhance vaccine efficacy.

誌謝 I
摘要 II
ABSTRACT III
目錄 IV
第一章 文獻回顧 1
1-1 前言 1
1-2 家禽流行性感冒病毒(禽流感病毒) 1
1-2-1 病毒型態、結構與分類 1
1-2-2 流感病毒表面醣蛋白的介紹 3
1-2-3 流感病毒流行病學 4
1-2-4 流感疫苗的開發與發展 5
1-3 桿狀病毒 11
1-3-1 桿狀病毒表現系統 12
1-3-2 桿狀病毒/哺乳動物細胞表現系統 13
1-3-3 桿狀病毒疫苗載體 15
1-4 FLP/FRT 重組酶系統 19
1-5 研究動機與目標 20
第二章 材料與方法 27
2-1 重組桿狀病毒之製備 27
2-2-1 BAC-TO-BAC®轉殖載體(DONOR PLASMID)之建構 27
2-2-2 重組表現載體之轉置反應(BAC-TO-BAC系統) 30
2-2-3 重組BACMID之分離 30
2-2-4 重組BACMID之轉染反應(TRANSFECTION) (製備P0病毒) 31
2-2-5 重組桿狀病毒的放大 31
2-2-6 單株重組桿狀病毒之篩選 32
2-2-7 濃縮重組桿狀病毒 33
2-2-8 重組桿狀病毒之感染效價分析(INFECTIOUS TITER) 33
2-2 細胞培養 34
2-2-1 昆蟲細胞培養 34
2-2-2 哺乳動物細胞培養 34
2-3 桿狀病毒轉導與分析 35
2-3-1 轉導哺乳動物細胞 35
2-3-2 流式細胞儀分析(FLOW CYTOMETRY) 35
2-3-3 細胞毒性分析(WST-1 ASSAY) 35
2-3-4 SDS-PAGE凝膠電泳、NATIVE-PAGE凝膠電泳及西方點墨法(WESTERN BLOT ANALYSIS) 36
2-3-5 BRADFORD蛋白質濃度測定 39
2-3-6 免疫螢光染色(IMMUNOFLUORESCENCE LABELING) 39
2-4 小鼠免疫試驗 40
2-5 小鼠血清免疫分析 41
2-6 酵素免疫墨點檢測法(ENZYME-LINKED IMMUNOSORBENT SPOT, ELISPOT) 44
第三章 結果與討論 50
3-1 FLPO/FRT基因延長表現系統 50
3-1-1 評估FLPO/FRT系統於肌肉細胞內的重組效率 51
3-1-2 評估表現FLPO重組酶對小鼠肌肉細胞的毒性 52
3-2-1 重組桿狀病毒的建構 53
3-2-2 以FLPO/FRT長效系統於哺乳動物細胞內的HA表現 56
3-2-3 篩選單株病毒以增加轉導細胞後的HA表現量 58
3-2-4 以單株桿狀病毒轉導細胞觀察HA蛋白的長期表現 59
3-2-5 分泌型HA蛋白三聚體結構的確認 60
3-3 小鼠免疫實驗 61
 新型桿狀病毒禽流感載體疫苗引發的小鼠免疫性 62
3-4 討論 62
第四章 未來展望 76
4-1 新型桿狀病毒載體的改良 76
4-2 記憶性免疫反應的探討 76
4-3 疫苗施打策略 77
第五章 參考文獻 79

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劉文慶,“桿狀病毒快速效價定量細胞之開發”, 碩士論文, 2013.