臺灣博碩士論文加值系統

在2012年9月，世界衛生組織 (WHO) 發現中東呼吸道症候群冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV) 首起感染案例，並且在2015年傳播至南韓與中國大陸，造成大流行。MERS-CoV為一種新型的冠狀病毒，具有外套膜且遺傳物質為單股正股RNA，此病毒與嚴重急性呼吸道症候群冠狀病毒 (severe acute respiratory syndrome coronavirus, SARS-CoV) 同屬beta亞科，但致死率遠高於SARS-CoV，且目前並無有效藥物或疫苗可以治療，本論文針對MERS-CoV之入侵宿主機制中扮演重要角色的棘蛋白 (spike protein, SP) 上的S2區域進行結構分析。MERS-CoV上的 spike protein可分為S1與S2兩部分，當病毒入侵宿主時會藉由S1上之RBD位置與宿主細胞表面上的受體結合，之後再利用S2上之HR1與HR2結合，形成six-helix bundle結構，此結構能幫助MERS-CoV病毒順利與宿主細胞膜進行融合，我們因此針對S2區段開發中和性單株抗體，希望能藉由此抗體去阻斷MERS-CoV入侵宿主之過程。S2抗體的制備流程可分為：(1) 對S2上HR1區域，根據抗原預測軟體分析其抗原性與表面性，並且避開可能有後修飾的位點，挑選表面抗原之可能序列以合成胜肽，並進行小鼠免疫；(2) 利用桿狀病毒表現系統製備S2及HR1之重組蛋白，同樣進行小鼠免疫。得到專一性單株抗體之後，進行抗體中和性測試，將HEK293T細胞轉染並表現帶有spike protein及EGFP的質體，再與含DPP4 receptor之Huh 7細胞共培養，觀察胞合體的生成。發現若加入以上製備各種抗體後，胞合體之生成比例減少，證實了抗體可藉由結合到S2區域，抑制病毒經膜融合進入宿主。預期未來將可應用在防疫或是流感病毒之研究工具。

In 2012, an outbreak in Saudi Arabia caused by the Middle East respiratory syndrome coronavirus (MERS-CoV) was reported by WHO. This novel coronavirus caused high fatality rates up to 36%, and was further spread to South Korea in 2015. Some researchers suggest that dromedary camels are the major reservoir host for MERS-CoV, and eventually cause human-to-human infections in health care settings. However, the actual role of dromedary camels in transmission of MERS-CoV is still unknown, and there is no specific antiviral drug against this virus. In this study, we focused on the spike protein (SP) on the virus surface and try to develop antibodies to inhibit the virus infection. The structure of spike protein contains two subunits, S1 and S2. The S1 subunit forms the globular head and binds to the host cell receptor DPP4 (dipeptidyl peptidase-4) with its receptor binding domain (RBD). Subsequently, the S2 subunit causes the conformation change to HR1/HR2, forming a six-helix bundle (6-HB) structure, and then insert its fusion peptide to the host membrane. Accordingly, S2 and HR1 is a good target to develop therapeutic antibodies against MERS-CoV. We firstly constructed and expressed S2 and HR1 domain in Bac-to-Bac expression system for the immunization of mice to produce antiserum (pAb). The mice were then sacrificed and their spleen cells collected for cell fusion with myeloma. The hybridoma cells producing specific monoclonal antibodies (mAb) were selected by enzyme-linked immunosorbent assay (ELISA). Both the pAb and mAb were successfully tested by neutralizing assays to confirm their capacity in inhibiting the invasion of MERS-CoV into host cells through its spike protein.

目錄
中文摘要 iii
Abstract iv
縮寫表 v
第一章 緒論 1
1.1中東呼吸道症候群冠狀病毒 1
1.1.1 MERS-CoV 病毒之歷史背景 1
1.1.2 MERS-CoV 病毒之型態與結構 2
1.1.3 MERS-CoV 病毒之基因體 4
1.1.4 MERS-CoV 病毒之蛋白質功能簡介 5
1.1.5 MERS-CoV 病毒生活史 6
1.2MERS-CoV膜上之醣蛋白研究 9
1.2.1 MERS-CoV 膜上棘蛋白介紹 9
1.2.2 MERS-CoV six-helix bundle 結構與病毒進入宿主之關係 12
1.3 研究動機 17
第二章 材料與方法 18
2.1 MERS-CoV S2、HR1抗原製備 18
2.1.1 MERS-CoV HR1抗原決定位預測 18
2.1.2 MERS-CoV S2、HR1重組蛋白製備 19
2.2傳統抗血清及單株抗體之製備 23
2.2.1 小鼠免疫 23
2.2.2 小鼠尾尖採血 23
2.2.3 融合瘤製備 23
2.2.4 融合瘤之篩選 25
2.2.5 限數稀釋 25
2.2.6 單株抗體之純化 26
2.3細胞培養法 26
2.3.1動物細胞培養 26
2.3.2昆蟲細胞培養 27
2.3.3細胞冷凍法 27
2.3.4細胞解凍法 27
2.4免疫學方法 28
2.4.1酵素連結免疫分析法 28
2.4.2 Western blot 28
2.5 細胞株共培養胞合體 29
2.6 蛋白質體學技術 29
第三章 結果與討論 31
3.1 MERS-CoV單株抗體製備 31
3.1.1 抗原片段之挑選與製備 31
3.1.2 S2及HR1重組蛋白抗原製備 39
3.1.3 單株抗體製備 45
3.1.4 融合瘤與單株抗體篩選 47
3.2 MERS-CoV 抗體之中和性測試 59
3.2.1 將表現SP之細胞與帶有DPP4受體之宿主細胞共培養 59
3.2.2 加入MERS-spike 抗體抑制 HES293T-SP-EGFP細胞進入帶有DPP4 之 Huh 7形成胞合體 62
第四章 未來研究方向 65
參考文獻 66

1.de Wit, E., et al., SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol, 2016. 14(8): p. 523-34.
2.Hijawi, B., et al., Novel coronavirus infections in Jordan. EMHJ, 2013. 19(Suppl. 1): p. S12-S18.
3.de Groot, R.J., et al., Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J Virol, 2013. 87(14): p. 7790-2.
4.WHO. Coronavirus infections: disease outbreak news. 2017; Available from: http://www.who.int/emergencies/mers-cov/en/.
5.Graham, R.L., E.F. Donaldson, and R.S. Baric, A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol, 2013. 11(12): p. 836-48.
6.Haagmans, B.L., et al., Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. The Lancet Infectious Diseases, 2014. 14(2): p. 140-145.
7.Azhar, E.I., et al., Evidence for camel-to-human transmission of MERS coronavirus. N Engl J Med, 2014. 370(26): p. 2499-505.
8.Hemida, M.G., et al., MERS coronavirus in dromedary camel herd, Saudi Arabia. Emerg Infect Dis, 2014. 20(7): p. 1231-4.
9.Reusken, C.B.E.M., et al., Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. The Lancet Infectious Diseases, 2013. 13(10): p. 859-866.
10.Alagaili, A.N., et al., Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia. MBio, 2014. 5(2): p. e00884-14.
11.van den Brand, J.M., S.L. Smits, and B.L. Haagmans, Pathogenesis of Middle East respiratory syndrome coronavirus. J Pathol, 2015. 235(2): p. 175-84.
12.Zumla, A., et al., Coronaviruses - drug discovery and therapeutic options. Nat Rev Drug Discov, 2016. 15(5): p. 327-47.
13.Neuman, B.W., et al., Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy. J Virol, 2006. 80(16): p. 7918-28.
14.Fehr, A.R. and S. Perlman, Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol, 2015. 1282: p. 1-23.
15.Mackay, I.M. and K.E. Arden, Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res, 2015. 202: p. 60-88.
16.Wang, Q., et al., MERS-CoV spike protein: Targets for vaccines and therapeutics. Antiviral Res, 2016. 133: p. 165-77.
17.Nieto-Torres, J.L., et al., Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog, 2014. 10(5): p. e1004077.
18.GODET, M., et al., TGEV corona virus ORF4 encodes a membrane protein that is incorporated into virions. VIROLOGY, 1992. 188: p. 666-675.
19.Neuman, B.W., et al., A structural analysis of M protein in coronavirus assembly and morphology. J Struct Biol, 2011. 174(1): p. 11-22.
20.KUBO, H., Y.K. YAMADA, and F. TAGUCHI, Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. VIROLOGY, 1994. 68(9): p. 5403-5410.
21.Cheng, P.K.C., et al., Viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome. The Lancet, 2004. 363(9422): p. 1699-1700.
22.Bosch, B.J., et al., The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex. Journal of Virology, 2003. 77(16): p. 8801-8811.
23.Pasternak, A.O., W.J. Spaan, and E.J. Snijder, Nidovirus transcription: how to make sense...? J Gen Virol, 2006. 87(Pt 6): p. 1403-21.
24.Perlman, S. and J. Netland, Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol, 2009. 7(6): p. 439-50.
25.Fung, T.S. and D.X. Liu, Coronavirus infection, ER stress, apoptosis and innate immunity. Front Microbiol, 2014. 5: p. 296.
26.Krijnse-Locker, J., et al., Characterization of the budding compartment of mouse hepatitis virus, Evidence that transport from the RER to the golgi complex requires only one vesicular transport step. J Cell Biol., 1994. 124(1-2): p. 55-70.
27.de Haan, C.A.M. and P.J.M. Rottier, Molecular Interactions in the Assembly of Coronaviruses. 2005. 64: p. 165-230.
28.Barlan, A., et al., Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection. J Virol, 2014. 88(9): p. 4953-61.
29.Millet, J.K. and G.R. Whittaker, Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. PNAS, 2014. 111(42): p. 15214-15219.
30.Du, L., et al., Identification of a receptor-binding domain in the S protein of the novel human coronavirus Middle East respiratory syndrome coronavirus as an essential target for vaccine development. J Virol, 2013. 87(17): p. 9939-42.
31.Lu, G., et al., Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature, 2013. 500(7461): p. 227-31.
32.Mou, H., et al., The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol, 2013. 87(16): p. 9379-83.
33.Wang, N., et al., Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res, 2013. 23(8): p. 986-93.
34.Li, F., et al., Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor. Science, 2005. 309(5742): p. 1864-1868.
35.Lu, L., et al., Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat Commun, 2014. 5: p. 3067.
36.Lu, G., Q. Wang, and G.F. Gao, Bat-to-human: spike features determining ''host jump'' of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol, 2015. 23(8): p. 468-78.
37.Belouzard, S., et al., Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses, 2012. 4(6): p. 1011-33.
38.Gao, J., et al., Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus. J Virol, 2013. 87(24): p. 13134-40.
39.Deng, Y., et al., Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein. Structure, 2006. 14(5): p. 889-99.
40.Xu, Y., et al., Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem, 2004. 279(47): p. 49414-9.
41.Supekar, V., et al., Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein. PNAS, 2004. 101(52): p. 17958–17963.
42.Xu, Y., et al., Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core. J Biol Chem, 2004. 279(29): p. 30514-22.
43.Liu, S., et al., Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. The Lancet, 2004. 363(9413): p. 938-947.
44.Liu, S., et al., Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. J Biol Chem, 2005. 280(12): p. 11259-73.