摘要 i
Abstract iii
目錄 v
表目錄 ix
圖目錄 x
壹、引言 1
一、伊波拉病毒歷史背景介紹 1
二、伊波拉病毒的生物特性 2
三、桿狀病毒表現系統基本背景介紹 3
四、桿狀病毒表現系統表現原理 4
五、桿狀病毒表面抗原呈現技術之原理及應用 5
貳、研究目的 8
參、材料與方法 9
一、實驗材料 9
（一）桿狀病毒表現系統 9
1. 線狀同源重組系統 9
2. 質體 10
3. 細胞培養 11
（二）藥品與試劑 13
1. 一般藥品與耗材 13
2. 市售套組 15
3. 培養基 16
（三）器材設備 17
二、實驗方法 18
（一）病毒重組轉移質體建構 18
1. PCR反應 19
2. PCR增幅反應 19
3. PCR反應條件 20
4. DNA瓊脂膠體電泳 20
5. 質體接合 21
6. 質體轉型 22
7. Colony PCR 22
8. 抽取質體 23
9. 核酸定序 24
（二）生產GP-Bac重組桿狀病毒 24
1. 共轉染實驗 25
2. 終點稀釋法 (End-point dilution) 25
3. 病毒效價測試 26
4. 病毒增殖 28
5. 病毒純化 28
6. 電子顯微鏡觀察 30
7. 共軛焦顯微鏡觀察 31
（三）伊波拉醣蛋白萃取與純化 32
1. 病毒感染，細胞收集與破碎 32
2. 伊波拉醣蛋白重力純化與透析 33
3. 伊波拉醣蛋白濃縮與定量 34
（四）老鼠免疫試驗 37
1. 重組病毒及伊波拉醣蛋白之抗原接種 37
2. 多株抗體效價測試 38
（五）GP-Bac假性病毒測試 39
1. 假性病毒測試於Sf21細胞 39
2. 假性病毒測試於Vero E6細胞 39
（六）西方墨點法 40
1. 蛋白質轉漬 (Transfer) 40
2. 抗體使用 41
3. 底片呈色 41
肆、實驗結果 43
一、重組桿狀病毒之建立與生產 43
（一）轉移質體之構築 43
（二）單一重組病毒株篩選 43
（三）伊波拉GP表面呈現之確認 44
（四）病毒純化結果及表面呈現之確認 44
（五）膜蛋白純化結果 45
（六）伊波拉GP之特性分析結果 46
二、小鼠免疫結果 46
（一）以ELISA檢測小鼠血清抗體之結果 47
（二）以西方墨點法檢測小鼠血清之結果 47
三、重組桿狀病毒作為假性病毒之測試 48
（一）重組病毒之GP64阻擋試驗（昆蟲細胞） 48
（二）重組病毒之GP64阻擋試驗（哺乳類細胞） 49
四、老鼠血清之中和性測試 50
伍、討論 52
陸、結論 55
柒、參考資料 83

表一、本實驗所使用之伊波拉病毒及馬堡病毒株 56
表二、引子序列清單 57

圖一、以重組桿狀病毒生產抗體之流程圖 58
圖二、各伊波拉轉移質體圖 59
圖三、於螢光顯微鏡下觀察到mCherry報導基因的表現 60
圖四、單一重組病毒株的篩選流程圖 61
圖五、各種伊波拉GP的單一GP-Bac重組桿狀病毒株感染Sf21細胞之西方墨點法結果 62
圖六、在相同感染條件下之單一GP-Bac病毒株 63
圖七、各GP-Bac以相同感染條件感染Sf21細胞後之西方墨點法結果 64
圖八、各伊波拉GP表現於感染的Sf21細胞表面 66
圖九、GP-Bac經蔗糖濃度梯度濃縮之結果 68
圖十、SUDGP及TAFGP膜蛋白純化過程和結果 69
圖十一、非變性SUDGP及TAFGP之西方墨點法結果 70
圖十二、GP蛋白之醣基化確認 71
圖十三、老鼠免疫排程及週數 72
圖十四、小鼠血清抗體ELISA測試 73
圖十五、小鼠血清抗體西方墨點法確認 74
圖十六、小鼠血清SUD2及TGP5對其他GPs之交互作用 75
圖十七、GP64阻斷的GP-Bac感染於昆蟲細胞 76
圖十八、GP-Bac假性病毒測試於哺乳類細胞 78
圖十九、其餘GP-Bac假性病毒測試於哺乳類細胞 80
圖二十、小鼠血清對重組病毒轉導效率之影響結果 82

1. Alvarez, C.P., Lasala, F., Carrillo, J., Muñiz, O., Corbí, A.L., and Delgado, R. (2002). C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 76, 6841–6844.
2. Bharat, T.A.M., Noda, T., Riches, J.D., Kraehling, V., Kolesnikova, L., Becker, S., Kawaoka, Y., and Briggs, J.A.G. (2012). Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci U. S. A. 109, 4275–4280.
3. Bornholdt, Z.A., Noda, T., Abelson, D.M., Halfmann, P., Wood, M.R., Kawaoka, Y., and Saphire, E.O. (2013). Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle. Cell 154, 763–774.
4. Carette, J.E., Raaben, M., Wong, A.C., Herbert, A.S., Obernosterer, G., Mulherkar, N., Kuehne, A.I., Kranzusch, P.J., Griffin, A.M., Ruthel, G., et al. (2011). Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343.
5. Chandran, K., Sullivan, N.J., Felbor, U., Whelan, S.P., and Cunningham, J.M. (2005). Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645.
6. Chang, T.-H., Kubota, T., Matsuoka, M., Jones, S., Bradfute, S.B., Bray, M., and Ozato, K. (2009). Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO modification machinery. PLoS Pathog 5, e1000493.
7. Chen, G.Y., Chen, C.Y., Chang, M.D.T., and et al. (2009). Concanavalin A affinity chromatography for efficient baculovirus purification. Biotechnol prog 6, 1669-1677.
8. Chen, Y.R., Wu, C.Y., Lee, S.T., Wu, Y.J., Lo, C.F., Tsai, M.F., and Wang, C.H. (2008). Genomic and host range studies of Maruca vitrata nucleopolyhedrovirus. J Gen Virol 89, 2315–2330.
9. Feldmann, H., Jones, S., Klenk, H.-D., and Schnittler, H.-J. (2003). Ebola virus: from discovery to vaccine. Nat Rev Immunol 3, 677–685.
10. Haase, S., Ferrelli, L., Pidre, M.L., and et al. (2013). Genetic engineering of baculoviruses. Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications, Prof. Victor Romanowski (Ed.), InTech, DOI: 10.5772/56976.
11. Jeffers, S.A., Sanders, D.A., and Sanchez, A. (2002). Covalent modifications of the ebola virus glycoprotein. J Virol 76, 12463–12472.
12. Kondratowicz, A.S., Lennemann, N.J., Sinn, P.L., Davey, R.A., Hunt, C.L., Moller-Tank, S., Meyerholz, D.K., Rennert, P., Mullins, R.F., Brindley, M., et al. (2011). T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proc Natl Acad Sci U S A 108, 8426–8431.
13. Kost, T.A., Condreay, J.P., and Jarvis, D.L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575.
14. Laakkonen, J.P., Mäkelä, A.R., Kakkonen, E., Turkki, P., Kukkonen, S., Peränen, J., Ylä-Herttuala, S., Airenne, K.J., Oker-Blom, C., Vihinen-Ranta, M., et al. (2009). Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells. PLoS ONE 4, e5093.
15. Lee, J.E., and Saphire, E.O. (2009). Ebolavirus glycoprotein structure and mechanism of entry. Future Virol 4, 621–635.
16. Lennemann, N.J., Walkner, M., Berkebile, A.R., Patel, N., and Maury, W. (2015). The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2. J Infect Dis 212 Suppl 2, S204–S209.
17. Leroy, E.M., Rouquet, P., Formenty, P., Souquière, S., Kilbourne, A., Froment, J.-M., Bermejo, M., Smit, S., Karesh, W., Swanepoel, R., et al. (2004). Multiple Ebola virus transmission events and rapid decline of central African wildlife. Science 303, 387–390.
18. Lin, W., Fan, H., Cheng, X., Ye, Y., Chen, X., and et al. (2011). A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice. Virology 8, 273.
19. Miller, L.K. (1988). Baculoviruses as gene expression vectors. Annu Rev Microbiol 42, 177–199.
20. Moller-Tank, S., and Maury, W. (2015). Ebola virus entry: a curious and complex series of events. PLoS Pathog 11, e1004731.
21. Oomens, A.G.P., and Wertz, G.W. (2004). The baculovirus GP64 protein mediates highly stable infectivity of a human respiratory syncytial virus lacking its homologous transmembrane glycoproteins. J Virol 78, 124–135.
22. Rhein, B.A., and Maury, W.J. (2015). Ebola virus entry into host cells: identifying therapeutic strategies. Curr Clin Microbiol Rep 2, 115–124.
23. Saeed, M.F., Kolokoltsov, A.A., Albrecht, T., and Davey, R.A. (2010). Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog 6, e1001110.
24. Sanchez, A., Kiley, M.P., Klenk, H.D., and Feldmann, H. (1992). Sequence analysis of the Marburg virus nucleoprotein gene: comparison to Ebola virus and other non-segmented negative-strand RNA viruses. J Gen Virol 73, 347–357.
25. Shurtleff, A.C., Warren, T.K., and Bavari, S. (2011). Nonhuman primates as models for the discovery and development of ebolavirus therapeutics. Expert Opin Drug Discov 6(3), 233-50.
26. Vigerust, D.J., and Shepherd, V.L. (2007). Virus glycosylation: role in virulence and immune interactions. Trends Microbiol 15, 211–218.
27. Volchkov, V.E., Feldmann, H., Volchkova, V.A., and Klenk, H.D. (1998). Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U. S. A. 95, 5762–5767.
28. Volchkov, V.E., Volchkova, V.A., Muhlberger, E., Kolesnikova, L.V., Weik, M., Dolnik, O., and Klenk, H.D. (2001). Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity. Science 291, 1965–1969.
29. Whelan, S.P., Barr, J.N., and Wertz, G.W. (2004). Transcription and replication of nonsegmented negative-strand RNA viruses. Curr Top Microbiol Immunol 283, 61-119.
30. WHO Ebola Response Team, Aylward, B., Barboza, P., Bawo, L., Bertherat, E., Bilivogui, P., Blake, I., Brennan, R., Briand, S., Chakauya, J.M., et al. (2014). Ebola virus disease in West Africa--the first 9 months of the epidemic and forward projections. N Engl J Med 371, 1481–1495.
Yang, Z.Y., Duckers, H.J., Sullivan, N.J., Sanchez, A., Nabel, E.G., and Nabel, G.J. (2000). Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat Med 6, 886–889.