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標題: | 利用質譜技術鑑定SARS冠狀病毒核殼蛋白主要抗原決定點 SARS-CoV Nucleocapsid Major Epitope Mapping by Mass Spectrometry |
作者: | Yu-Tsung Chen 陳昱璁 |
指導教授: | 周綠蘋 |
關鍵字: | 嚴重急性呼吸道症候群冠狀病毒,抗原決定點, SARS-CoV,epitope, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 2003年3月,世界衛生組織 (WHO) 對全球發出警告,有一種未知病原的非典型肺炎,從越南、香港、中國等地區向全世界蔓延,並在同年3月正式命名為急性嚴重呼吸道症候群 (Severe Acute Respiratory Syndrome, SARS) ,至今造成8400多人感染以及奪走774條寶貴的生命,SARS自發病到死亡時間很短,且致病原為一種新型病毒,沒有有效疫苗以及治療藥物,因此有必要研究如何應對突如其來的感染疾病,並且儘快研發早期偵測試劑以及疫苗,為下一波SARS可能來襲作準備。SARS為新興的冠狀病毒 (coronavirus, CoV) ,雖然其基因與其他冠狀病毒少有相似,但同樣具有針狀蛋白質 (spike protein, S) 、膜蛋白 (membrane protein, M) 、套膜蛋白 (envelope, E) 以及核殼蛋白 (nucleocapsid, NC) 等結構的蛋白質。 本實驗室取得核殼蛋白之基因序列,並利用基因重組之技術以大腸桿菌 (E. coli) 大量製造重組之核殼蛋白,將所得之重組核殼蛋白利用西方墨點法(Western blot)技術篩檢病人血清,發現具有良好的抗原抗體反應,更進一步要瞭解核殼蛋白之主要免疫抗原點 (major epitopes) ,便將所純化的重組核殼蛋白利用酵素或是化學物質使其在特定胺基酸產生斷裂,所得之胜肽再利用高效液態層析儀 (High Performance Liquid Chromatography, HPLC) 分離,收集胜肽以質譜儀 (MALDI-TOF and ESI Q-TOF) 分析鑑定,並利用dot-blot assay將所得的胜肽與針對核殼蛋白有特異性之兔子多株抗體先進行辨認,篩選抗體可以辨認的胜肽,再利用ELISA將這些胜肽進行病人偵測,藉此了解每個病人辨認區域並分析共有的免疫抗原點,即主要抗原決定點。 本次實驗在兔子血清辨認方面篩選出24抗體可辨認的胜肽,利用病人血清篩選的結果找到一個胜肽L26 (H301-K339),在十二位病人血清的檢測下,有十一位病人血清會對這個胜肽產生辨認而一般人血清則沒有反應,因此我們找到一個主要抗原決定點位在胜肽L26上,而利用所找到的這個胜肽,將有機會開發較完整蛋白特異性更高、䀱陽性更低的偵測試劑。而本實驗這樣的方法亦可提供其他傳染疾病,或是類似於SARS這樣突如其來的危險疾病之篩檢以及治療、偵測方式之快速開發。 In March 2003, a Global Alert to an unknown disease was launched by WHO. This unknown disease cause severe atypical pneumonia and was spread out from mainland China, Hong Kong and Vietnam to the whole world. This new disease, named severe acute respiratory syndrome (SARS) cause over 8,400 people infected and 774 people died. In April, a new coronavirus, SARS-CoV was proven to be the causative agent, and still had no vaccine and no treatment. In order to prevent SARS recurrence, searching for vaccine design and diagnosis method is important. The genomic organization of SARS-CoV is similar to those of other known coronavirus. However, sequences alignment indicates that SARS-CoV does not closely resemble any of the previously characterized coronavirus. SARS-CoV contains open reading frames (ORF) for four structural proteins: nucleocapsid (NC), spike (S), envelope (E), and membrane (M) protein. The nucleocapsid (NC) gene of SARS virus was constructed and ligated with pQE-30 expression vector. Then, rNC protein was expressed in E. coli and purified by nickel column. The immunoreactivity of the rNC protein was confirmed by Western blot with SARS patients’ sera. In order to obtain the major epitopes, first of all, the rNC protein was digested with site-specific protease. Second, the digested fragments were separated and collected by HPLC and further confirmed by dot-blot assay and ELISA with rabbit polyclonal anti-serum and SARS patients’ sera. Finally, these candidate epitopes were identified by tandem mass spectrometry. In conclusion, 24 candidate epitopes are recognized by rabbit polyclonal anti-sera and the major epitope L26 (H301-K339) is confirmed by 12 SARS patients’ sera. The major epitope we found in this study may be contributed to early detection of SARS-CoV. Using the major epitope rather than intact protein for diagnosis may increase the specificity and reduce false-positive rate. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35964 |
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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