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標題: | 製備中東呼吸道症候群冠狀病毒之中和性抗體 Development of Neutralizing Antibodies against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) |
作者: | Ying-Syuan Li 李盈萱 |
指導教授: | 莊榮輝 |
關鍵字: | 中東呼吸道症候群冠狀病毒,抗體, Middle East Respiratory Syndrome Coronavirus,Antibodies, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67077 |
DOI: | 10.6342/NTU201702976 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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