輔仁大學
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學校名稱輔仁大學
系所名稱生命科學系
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研究生(中)李秉勳
研究生(英)Ping-Hsung Lee
論文名稱(中)由重組桿狀病毒感染之 Sf 9 細胞株中表現及純化嚴重急性呼吸道症候群冠狀病毒之 S547 刺突蛋白片段並建立一個以細胞為基礎之抗病毒藥物篩選模式
論文名稱(英)Expression and Purification of Severe Acute Respiratory Syndrome-Coronavirus Spike Protein S547 from Recombinant Baculovirus Infected Sf9 Cells and Establishment a Cell-Based Model for Anti-viral Drugs Screening
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指導教授(中)郭育綺
指導教授(英)Yuh-Chi Kuo
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關鍵字(中)嚴重急性呼吸道症候群 呼吸道 病原體 墨點法 類固醇 染色法
關鍵字(英)SARS Spike future system China data
摘要(中)2002年一新興呼吸道疾病-嚴重急性呼吸道症候群 (Severe Acute Respiratory Syndrome;SARS) 在短時間內於全世界發生,造成全球8,098 個疑似病例其中有774個死亡病例。經各國通力合作,證實造成 SARS 的病原體為一全新之冠狀病毒 (Coronavirus),因此 WHO將此病毒正式命名為 SARS-Coronavirus (SARS-CoV)。SARS 流行期間主要採用三氮唑核? (ribavirin) 和皮質類固醇 (corticosteroid) 共同使用的治療方式,然而其卻無顯著治療效果,為避免 SARS 再次傳播固需要有效治療 SARS 藥物。SARS 傳播主要藉由空氣傳染途徑,以病毒刺突蛋白 (spike) 與宿主細胞表面之受器血管緊張素轉換?2 (Angiotensin converting enzyme 2;ACE2) 結合而染感宿主。本實驗最終目的為希望利用 SARS-CoV 的spike 與 ACE2 間結合關係,以建構一個以細胞為基礎之安全藥物篩選平台。為了獲得 spike 蛋白質片段,於研究中利用重組桿狀病毒感染Sf9 細胞,以獲得由第16 個氨基酸至第547 氨基酸序列之spike蛋白片段,特稱為 S547,並利用西方墨點法確認其大小約為 85 kDa。此外,針對 S547 上由六個組氨酸所組成之標誌 (His-Tag),以 Ni2+-NTA 純化系統加以純化,並以50 mM 至90 mM 間各不同濃度之 imidazole 去除非專一性蛋白以加強純化效果。為了確認 ACE2在細胞株之表現,實驗中以Vero E6做為標的細胞,利用反轉錄連鎖聚合?反應、西方墨點法以及免疫螢光染色法分析,證明ACE2 可分佈與表現於 Vero E6 細胞表面。為了確認 S547可與 Vero E6 進行鍵結,以免疫螢光染色法證明 S547 蛋白確實會結合到 Vero E6 細胞上。為了建立安全之抗病毒藥物篩選平台,初步以 Vero E6培養於96孔培養盤中,加入 S547之後以Anti-His Tag monoclonal antibody 進行ELISA分析,結果發現 S547 在此以細胞為基礎之模式中,確實能與 Vero E6 上所表現之 ACE2 結合。未來,當成熟建立此模式後,將應用於抗病毒藥物之篩選。
摘要(英)In 2002, a new contagious disease had been found in Southern China and the disease spread widely and resulted in 8098 cases and 774 deaths. This disease was characterized as an atypical pneumonia symptom. The World Health Organization defined it as severe acute respiratory syndrome (SARS). The pathogen of SARS is a new coronavirus and named as SARS-coronavirus (SARS-CoV). Combination of ribavirin and corticosteroid is the most frequent antiviral treatment for SARS. However, the unobvious curative and annoying side effects for these medicines, it is urgent to find new anti-SARS drugs. SARS-CoV spreads mainly via respiratory routes and infects host cells by binding of viral spike protein to cellular angiotensin converting enzyme-2 (ACE2). The aim of thesis is to express the spike protein fragment and establish an anti-viral drug screening model. In the present study, I have obtained a recombinant baculovirus that carried a cDNA encoding SARS-CoV spike proteins from 16 a.a. to 547 a.a. Then a histidine Tag fusion protein (85 kDa), named as S547, was purified from recombinant baculoviruses infected sf9 cells by the Ni2+-NTA system. The results of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting indicated that ACE2 expression could be detected in Vero E6 cells. The data of immunofluorescent staining demonstrated that S547 proteins can bind to Vero E6 cells. Moreover, I have set up the cell-based ELISA to prove that S547 could bind to Vero E6 cells in a dose-dependant manner. In the future, the cell-based ELISA model will be applied for screening anti-viral drugs.
論文目次目錄 頁 中英縮寫對照表 2 中文摘要 4 英文摘要 5 壹、前言 6 一、 嚴重急性呼吸道症候群 (severe acute respiratory syndrome) 之發生 6 二、 冠狀病毒與 SARS-CoV 8 三、 SARS-CoV 之S 蛋白與細胞受器間之關係 10 四、 SARS 治療策略 12 五、 研究動機與目的 13 貳、實驗材料與方法 15 一、 秋行軍蟲卵巢組織細胞 (sf9 cell line) 培養 15 二、 HeLa 與 Vero e6 細胞株培養 15 三、 攜帶 SARS-CoV 之 S547 片段重組桿狀病毒來源 15 四、 重組桿狀病毒擴增 16 五、 重組桿狀病毒濃度定量 16 六、 S547 融合蛋白質之生成 16 七、 S547融合蛋白質之製備與純化 17 八、 細胞質蛋白與細胞膜蛋白之製備 17 九、 蛋白質濃度測定 18 十、 硫酸十二酯鈉-聚丙烯醯胺膠體電泳法與西方墨點法 18 十一、全量細胞核糖核酸之製備 19 十二、反轉錄?聚合?連鎖反應 20 十三、細胞免疫螢光染色 21 十四、以細胞為基礎之酵素免疫分析 21 十五、藥物之製備 22 十六、統計學分析 23 ?、實驗結果 24 肆、討論 31 伍、參考文獻 37 陸、表 45 柒、圖 47 捌、附錄 64
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