請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21838
標題: | 中東呼吸道症候群冠狀病毒之棘蛋白、膜蛋白與包膜蛋白的表現與分離 Protein Expression and Isolation of the MERS-CoV Spike Protein, Membrane Protein and Envelope Protein |
作者: | AN-JIE TAN 譚安絜 |
指導教授: | 莊榮輝 |
關鍵字: | MERS-CoV冠狀病毒,Spike protein (S),Membrane protein (M),Envelope protein (E),桿狀病毒表現系統, Middle East respiratory syndrome coronavirus,Spike protein,Membrane protein,Envelope protein,Baculovirus expression system, |
出版年 : | 2018 |
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) 於2012年首度在沙烏地阿拉伯被發現,之後也陸續傳播到歐洲、亞洲、非洲及北美洲。截至2018年9月為止已經有2249例確診病例,其中有798名病患死亡,死亡率高達36%。然而目前尚無有效的疫苗與藥物可供治療,因此發展出有效預防的疫苗與治療的抗體對於防疫工作迫切重要。
MERS-CoV為含有單股正向RNA的冠狀病毒,其外套膜上具有三種結構蛋白spike (S)、membrane (M)及envelope (E),從感染時辨識宿主與受體結合,到病毒出芽時的組裝與結構穩定都和這三種結構蛋白息息相關,也是近年來重要疫苗候選蛋白與抗病毒藥物的主要標的。本研究致力於利用桿狀病毒與昆蟲細胞表現系統製備MERS-CoV的三種結構蛋白,以提供未來疫苗與抗體之研發需求。將含有S、M及E基因的重組Bacmid經轉染至Sf21昆蟲細胞後,已成功獲得能表現S、M及E重組蛋白之桿狀病毒,並進行了最適表現條件探討,成功分離各重組蛋白後再以質譜儀分析確認S、M及E重組蛋白之的胺基酸序列與由基因所推測之序列完全相符,完成製備疫苗與抗體前的抗原表現先導實驗。本研究亦利用大腸桿菌表現系統生產S蛋白質上負責與受體 dipeptidyl peptidase 4 (DPP4) 結合之receptor binding domain (RBD),並以實驗室先前製備之Anti-RBD單株抗體 (6-9B及2-11A) 來進行分析,實驗結果顯示RBD於大腸桿菌表現時會形成inclusion bodies。 MERS-CoV was first identified in Saudi Arabia in 2012 and has spread to Europe, Asia, Africa and North America. A total of 2,249 MERS-CoV-infected cases with a mortality of 36% was reported in September 2018. WHO declares that there are no vaccines or specific treatment available now. Thus, there is an urgent need for the development of vaccines and theranostic antibodies for better pandemic preparedness. MERS-CoV has a positive sense, single-stranded RNAs, including the spike (S), membrane (M) and envelope (E) genes. These three viral surface structure proteins are involved in the cell invasion, structural stability and the release of the viral particles, and can become potential vaccines or be major targets of anti-virus drugs. The present work was aimed to establish the MERS-CoV S, M, and E recombinant protein expression system for the demand of preparing vaccines and antibodies in the future. The recombinant baculovirus bacmids containing S, M, and E genes of MERS-CoV has been tansfected to the Sf21 cells, and the baculoviruses that can individually express recombinant S, M and E proteins were successfully produced. The amino acid sequences of the recombinant S, M and E proteins analyzed by mass spectrometry revealed that they are exactly the same as the sequences deduced from MERS-CoV S, M and E genes. The optimal protein expression conditions and isolationn procedures of MERS-CoV S, M and E proteins were also determined and investigated in the present study. In addition, the receptor binding domain (RBD) of S protein, which is responsible for binding to the receptor dipeptidyl peptidase 4 (DPP4), was expressed in Escherichia coli and was analyzed by western blotting using the previously prepared anti-RBD monoclonal antibody (6-9B and 2-11A) in the laboratory. The experimental results showed that RBD formed inclusion bodies while expressed in E. coli. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21838 |
DOI: | 10.6342/NTU201804332 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-1.pdf 目前未授權公開取用 | 2.96 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。