臺灣博碩士論文加值系統

嚴重急性呼吸道症候群 (Severe Acute Respiratory Syndrome, SARS)為一具有高度傳染性和高致死率的新興疾病，在2003年2月至7月間造成全球性的大流行，致病原目前已知為一新型變種冠狀病毒，命名為SARS-CoV。至2003年7月截止，總計感染人數8098人。由於目前對
於SARS並無有效的治療方法，為免SARS再次捲土重來爆發流行，因此疫苗的研發成為十分重要的工作。本論文使用重組腺病毒當做載體設計SARS疫苗，重組腺病毒能夠高量的表達蛋白質，而且感染不會引起嚴重危害，使用上具有高安全性，目前已被廣泛拿來試驗當作疫苗的可行性。以重組腺病毒為載體之疫苗在許多病毒性疾病中幾乎都能夠引發抗體反應產生中和抗體，在某些病毒性疾病還可額外引發細胞免疫。另外，重組腺病毒因為能感染黏膜組織，所以能夠引發黏膜性免疫（mucosal immunity），對於藉由性接觸或黏膜感染的病毒疾病，提供了有效的保護效果。本論文以SARS-CoV結構蛋白基因spike(S), membrane(M), envelope(E) 為基礎建構重組腺病毒疫苗，目前已建構出三種重組腺病毒，分別為攜帶S基因、帶有M和E基因和帶有S,M,E基因的三種重組腺病毒載體。載體建構完成後，以質體DNA和重組腺病毒當做疫苗施打BALB/c小鼠，分別以施打DNA疫苗、施打重組腺病毒疫苗和先施打DNA疫苗再追加重組腺病毒疫苗的方式在小鼠模式中比較以不同施打策略誘發之免疫反應。最後，本論文將利用ELISA方法偵測免疫後小鼠體內有無引發SARS病毒專一性抗體。

The Severe acute respiratory syndrome (SARS) is a newly emerged disease with high infectivity and morbidity. It caused the global pandemic between February and July in 2003. As of July 31, 2003, SARS has been affecting 8098 individuals and causing 774 deaths (WHO). The causative agent for SARS has been identified as a novel coronavirus, the SARS-CoV. There are no effective therapies for SARS at present and it is very important to prevent the reemergence of SARS. Among the viral vectors used in vaccine design, recombinant adenovirus is known for high-level protein expression and efficient induction of neutralizing antibody and/or cellular immunity in many virus diseases, yet only causes mild illness in humans. Besides, recombinant adenovirus vaccine can induce mucosal immunity, which can effectively protect host from viral diseases disseminated through mucosa. In this study, we used the recombinant adenovirus as vactor and the three SARS structure proteins, spike (S), membrane (M) and envelope (E) as targets, to develop SARS vaccines. We have constructed three recombinant adenoviral vectors encoding S (Adv/S), M and E (Adv/ME) or S, M and E (Adv/SME), respectively. The pShuttle plasmids encoding the M/E and S, were used as DNA vaccines. Three immunization strategies, DNA immunization, ADV vaccination and DNA/ADV prime-boost immunization regimen were performed in mice to compare their efficiency in inducing humoral and cellular immune responses. The SARS-CoV specific antibody induced by the recombinant adenovirus vaccines and DNA vaccine will be determined by ELISA.

目 錄 　　　　　　　1

圖表目錄 　3

中文摘要 4

英文摘要 5

第一章、緒論 6
1.1 SARS病毒簡介 6
1.1.1嚴重急性呼吸道症候群病毒之流行病學 6
1.1.2 SARS病毒之鑑定與分類 7
1.1.3病毒基因體結構 8
1.1.4 SARS病毒結構蛋白 　　 　　　　 8
1.2 疫苗的發展 11
1.2.1 DNA疫苗 11
1.2.2重組腺病毒疫苗 13
1.2.3 SARS疫苗的研發 16
1.3 研究目的和實驗設計 17

第二章、材料與方法 18
2.1 實驗材料 　　　　18
2.2 實驗方法 23
2.2.1細胞株與細胞培養 24
2.2.2重組腺病毒的製備 24
2.2.3 以大腸桿菌(BL21)表現S、M、E蛋白　　　　 30
2.2.4 硫酸十二酯鈉多醯胺凝膠電泳 31
2.2.5 小鼠免疫實驗 33
2.2.6酵素連接免疫吸附分析法（ELISA） 　 34

第三章、結果 35
3.1 重組腺病毒疫苗之構築 　　 35
3.2 蛋白質表現 36
3.3 小鼠免疫實驗 　　　37
3.4　測定抗SARS病毒S、M、E蛋白質之特異性IgG 37

第四章、討論 38

圖表 41
附錄 61
參考文獻　　　 　　 　　　　 63

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