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研究生:徐偉婷
研究生(外文):HSU, WEI-TING
論文名稱:以桿狀病毒表現載體系統發展冠狀病毒的抗體檢測及疫苗同步產生平台
論文名稱(外文):Development of Coronavirus Antibody Detection and Vaccine Simultaneous Production Platform by Baculovirus Expression Vector System
指導教授:趙裕展
指導教授(外文):Chao, Yu-Chan
口試委員:林昌棋張惠雯顏莉蓁吳岳隆
口試委員(外文):LIN, CHANG-CHICHANG, HUI-WENYEN, LI-CHENHU, YUEH-LUNG
口試日期:2021-04-16
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:141
中文關鍵詞:桿狀病毒載體表現系統冠狀病毒抗體檢測疫苗
外文關鍵詞:Baculovirus expression vector systemCoronavirusAntibody detectionVaccine
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冠狀病毒是一種可感染多種物種並廣泛存在於不同動物體內的病毒。其中豬流行性下痢病毒(PEDV),即是一種以豬隻為主要宿主的冠狀病毒,其感染範圍遍及全球,當豬隻感染此病毒後會引起嚴重下痢至死。本研究利用桿狀病毒表現載體系統發展豬下痢病毒抗體檢測及疫苗同步產生平台。透過我們所研發的桿狀病毒表面呈現系統,建構PEDV的棘蛋白(spike)重組病毒 (PEDV-S-6MC-Bac),表現PEDV 2b(G2b)強毒流行株的全長棘蛋白於昆蟲細胞表面,並以此建立cell-based ELISA系統。我們利用此系統中的昆蟲細胞上完整展示的三聚體棘蛋白進行豬下痢病毒血清學檢測共100個豬血清樣品,其結果達到100%的敏感性,97%的特異性,以及與免疫細胞化學染色結果高度的一致性(???? = 0.98)。另外,在疫苗系統的開發中,我們使用肌肉注射PEDV-S-FL-Bac的方式來免疫小鼠。與對照組相比,免疫PEDV-S-FL-Bac組別的血清中的抗豬下痢病毒特異性IgG的抗體力價顯著增加。為了進一步測試我們的桿狀病毒檢測與疫苗整合系統,我們也使用了導致2019新冠肺炎(COVID-19)全球致死性大流行之冠狀病毒:嚴重急性呼吸系統綜合症冠狀病毒2(SARS-CoV-2)作為測試模式。並利用上述之桿狀病毒表現系統,分別展示SARS-CoV-2的核殼蛋白(N)和棘蛋白(S)在昆蟲細胞上並用來精確檢測患者的血清。值得注意的是,儘管許多患者的抗S抗體被我們的cell-based ELISA平台檢測到,但幾乎無法在Western blot中偵測到。這結果顯示,COVID-19患者可能主要產生辨認完整構型的抗S抗體。此外,相同的桿狀病毒構建體可在桿狀病毒包膜上展示核殼蛋白(SARS-CoV-2-N-Bac)或棘蛋白(SARS-CoV-2-S-Bac),以作為載體疫苗。動物實驗結果顯示,以SARS-CoV-2-N-Bac或SARS-CoV-2-S-Bac為疫苗施打小鼠可引起強烈特異性的抗體反應,而在無佐劑的狀況下,SARS-CoV-2-S-Bac還可誘導出極為有效的中和性抗體。我們的重組桿狀病毒表現系統可展示抗原並維持良好的抗原性與多聚合立體結構,並刺激中和性抗體的產生以利血清檢測。綜合以上結果,透過桿狀病毒表現系統所發展的冠狀病毒的抗體檢測及疫苗同步產生平台,可作為一種可靠及有效的疫苗與血清檢測系統,而這系統對豬下痢病毒及新冠病毒的感染尤其有效。此重組桿狀病毒所建立的檢測及疫苗系統為一種安全且易於操作的平台,期望將來可成為對抗其他新興和再復發病毒的立即高效之疾病控制系統。
Coronaviruses are diverse groups of viruses infecting many different animals. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. How to efficiently detect the PEDV infection and produce effective vaccines are the key to combat this disease. In this study, we develop an integrated system for the detection and vaccination of PEDV. We constructed a recombinant baculovirus (PEDV-S-6MC-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain. First, we used PEDV-S-6MC-Bac to generate a novel cell-based enzyme-linked immunosorbent assay (ELISA) for convenient PEDV antiserum detection. We analyzed 100 pig serum samples, and the cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement (???? = 0.98) with immunocytochemical staining results. Second, for vaccine development, we immunized mice with PEDV-S-6MC-Bac by intramuscular injection. Compared with the control group, PEDV-S-FL-Bac immunized group showed a significant increase in systemic anti-PEDV S-specific IgG. For further application of this novel integrated detection and vaccination platform, we used another coronavirus: the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for a separate test. This is the virus which causes coronavirus disease 2019 (COVID-19) lethal pandemic over the world. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based detection system to detect patient serum with accuracy. Notably, anti- SARS-CoV-2-S antibodies from many patients, although well-recognized by our newly developed detection system with SARS-CoV-2 spike displaying insect cells, were barely detectable by Western blot. This provides evidence that COVID-19 patients primarily produce conformation-dependent anti-SARS-CoV-2-S antibodies. Furthermore, the same baculovirus constructs can display SARS-CoV-2-N or SARS-CoV-2-S proteins on the baculovirus envelope and serve as vector vaccines. Animal experiments show that SARS-CoV-2-S-Bac or SARS-CoV-2-N-Bac immunization in mice elicited a strong and specific antibody response, and SARS-CoV-2-S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination. Our results demonstrated that our recombinant baculovirus system could serve as a safe and easy to manipulate antigen to produce the effective detection and vaccine against the virus infection. Moreover, the combination of detection and vaccination speeds up the fight against diseases, and therefore the system can be applied to the effective control and prevention of other emerging or recurring viral diseases in the future.
目錄......................................................................................................................................................... II
圖目錄...................................................................................................................................................... IX
表目錄...................................................................................................................................................... XII
中文摘要.................................................................................................................................................. XIII
ABSTRACT............................................................................................................................................... XV
I. Introduction.......................................................................................................................................... 1
II. Material and Methods.......................................................................................................................... 25
III. Results................................................................................................................................................ 43
IV. Discussion.......................................................................................................................................... 56
V. Figures and Tables............................................................................................................................... 67
VI. References.......................................................................................................................................... 100

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