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標題: | 建構MERS-CoV類病毒顆粒以誘使BALB/c小鼠產生中和性抗體 Generation of MERS-CoV virus-like particles to induce neutralizing antibodies in BALB/c mice |
作者: | Yu-Pin Yeh 葉妤玢 |
指導教授: | 張明富 |
關鍵字: | 中東呼吸道症候群,類病毒顆粒,中和性抗體, MERS-CoV,Virus-like particles,neutralizing antibodies, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 中東呼吸道症候群冠狀病毒 (Middle East respiratory syndrome coronavirus;MERS-CoV) 是於2012年的沙烏地阿拉伯地區首度被發現引起新的呼吸道疾病。隨著至中東旅遊的人數增加,此病毒也傳播至歐洲、亞洲、非洲及北美洲。截至今年的六月為止已經有1733名確診的案例,其中有628名患者死亡,死亡率高達36%,較致死率約9.6%的嚴重呼吸道症候群 (Severe acute respiratory syndrome;SARS) 更為嚴重。MERS-CoV是一具有單股正向RNA基因體的病毒,其結構蛋白質有spike、envelope、membrane及nucleocapsid。當MERS-CoV感染宿主細胞時,會利用外圍突起之Spike與宿主細胞表面的dipeptidyl peptidase 4 (DPP4) 結合,使細胞膜融合促使病毒得以進入細胞內。至目前為止,並沒有任何專一性的藥物及疫苗能夠治療MERS,多半是給予患者輔助性之療法。為了發展MERS-CoV疫苗,因此本研究利用昆蟲細胞同時表現MERS-CoV的三種結構蛋白質,分別為spike、envelope及membrane,進而組裝成MERS virus-like particles (MERS-VLPs)。藉由穿透式電子顯微鏡能夠觀察到形成的MERS-VLPs為直徑約100 nm之圓形顆粒,並且有類似Spike之棘狀突起。另外在免疫螢光染色的結果顯示MERS-VLPs與其受器DPP4有共位 (co-localization) 之情形,進一步證明所組裝之VLPs具有其病毒特性,且有spike蛋白質鑲嵌於膜外。由於VLPs能夠模擬病毒在真實環境下的結構,不具有複製能力且感染性低等優點,本篇研究利用MERS-VLPs作為抗原施打於BALB/c小鼠,結果證實MERS-VLPs可以使小鼠發生免疫反應而產生中和性抗體。未來可以利用MERS-VLPs產生humanized之抗體,希望能以被動免疫療法用於治療MERS。 The Middle East respiratory syndrome coronavirus (MERS-CoV) was initially identified in the Kingdom of Saudi Arabia in September 2012. The transmission of MERS-CoV have been reported from countries in Europe, Asia, Africa, and North-America. MERS-CoV has caused 1733 laboratory confirmed cases till June 2016 with a case-fatality rate of 36% which is much higher than the 9.6% mortality rate caused by severe acute respiratory syndrome coronavirus. MERS-CoV is a positive single-strand RNA virus, with structural proteins includes spike, membrane, envelope and nucleocapsid protein. MERS-CoV uses dipeptidyl peptidase 4 (DPP4) as its functional receptor. The spike protein initiates virus entry by binding to DPP4, the binding leads to membrane fusion. To date, no drugs and vaccines have been clinically approved to control MERS-CoV infection. One of the most promising tools in vaccine development over the last two decades is the use of recombinant-based virus-like particles (VLPs). VLPs are multiprotein structures closely resembling natural virions. VLPs that lack viral genetic materials are non-infectious and non-replicating particles. On the other hand, VLPs display intact and biochemically active antigens on their surface and thus, show high immunogenicity and antigenicity. Importantly, VLPs interact with the host immune system inducing humoral and cellular responses similarly to the native pathogens. In order to generate MERS-VLPs, high five cells were co-infected with recombinant baculoviruses expressing the viral structural proteins spike, envelope, and membrane. MERS-VLPs were harvested and purified by sucrose gradient centrifugation. Transmission electron microscopy detected the MERS-VLPs with diameters approximately 100 nm, and spike protein appeared around the spherical particles. Immunofluorescence assay demonstrated colocalization of the spike-incorporated VLPs with DPP4. In addition, BALB/c mice were vaccinated with the partially purified MERS-VLPs. MERS-VLPs induced host immune response and generated neutralizing antibodies. In the future, humanized antibodies could be generated as a promising passive immunotherapy for MERS. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49422 |
DOI: | 10.6342/NTU201603168 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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