臺灣博碩士論文加值系統

嚴重急性呼吸道症候群是一種新的傳染病，SARS冠狀病毒為導致該病的病原體。在我們實驗室先前的研究中，發現SARS冠狀病毒膜蛋白在變性處理的情況下，不易用西方點墨法偵測到。在本實驗中，我們利用西方點墨法與體外表達蛋白質(同位素標幟)系統，在蛋白質電泳上層膠發現經熱變性(不同加熱溫度，或不同加熱時間)的聚集膜蛋白；因此，我們認為熱會造成SARS-CoV膜蛋白的聚集，使得我們不易在蛋白質電泳下層膠偵測到。為了了解SARS-CoV膜蛋白因熱引起聚集的機制，我們試著用不同的denaturant(SDS、urea、Triton-X100)回溶聚集的SARS-CoV膜蛋白，並發現聚集的膜蛋白不易被回溶。另外，我們也發現膜蛋白疏水性區域：a.a.61-90、a.a.91-100、a.a.136-170對於膜蛋白的聚集是必須的；而且突變的SARS膜蛋白MΔ91-100，會在正常SARS-CoV膜蛋白的存在下發生聚集現象。這似乎意味著SARS-CoV膜蛋白因熱引起的聚集現象，是由於蛋白質疏水性區域分子之間的作用力。 這研究顯示，如果我們要分析SARS-CoV膜蛋白時，應避免一般的加熱步驟；也提供SARS-CoV對熱不穩定現象的可能解釋。SARS-CoV膜蛋白因熱引起的聚集也提供研究蛋白質聚集的一個模式。

Severe acute respiratory syndrome (SARS) is a new respiratory illness caused by the infection of a novel coronavirus called SARS-CoV. Previous studies in our laboratory found that SARS-CoV membrane protein could be easily detected using Western blotting in non-denaturing condition but not in regular denaturing condition. Using Western blotting analysis and in vitro transcription /translation system (isotope-labeled), the aggregated membrane protein with the thermal denaturation (different temperature or different boiling time) was detected in the stacking gel. Therefore, the thermal aggregation of SARS-CoV membrane protein results in the inefficiency in detecting the membrane proteins. The aggregated membrane protein could not be re-dissolved by different denaturants: 2%SDS, 6Murea, 1%Triton-X100 Furthermore, the hydrophobic regions with amino acid residues from 61 to 90, from 91 to 100, and from 136 to 170 are essential for the aggregation of membrane protein. The membrane mutant protein with deletion from a.a.91 to 100 could be aggregated with wild type membrane protein when induced by heat. These results indicate that aggregation of SARS-CoV membrane protein induced by heat is through extensive interaction in hydrophobic domains. Therefore, if SARS-Cov membrane protein needs to be analyzed using SDS-PAGE, regular boiling treatment should be avoiding. Thermal aggregation of SARS-CoV membrane protein may be also responsible for the inactivation of this virus by heat. Thermal aggregation of SARS-CoV membrane protein also provides a model to study the protein aggregation.

目 錄 中文摘要………………………………………………………………………1 英文摘要………………………………………………………………………2 序論………………………………………………………………………………3 實驗材料與方法………………………………………………………………………………8 實驗結果…………………………………………………………………………………….. 17 討論…………………………………………………………………………………………...22 參考文獻……………………………………………………………………………………...25 圖一…………………………………………………………………………………………...30 圖二…………………………………………………………........…………………………...31 圖三……………...……..……………………………………………………………………..32 圖四…………………………………………………………………………………………...33 圖五…………………………………………………..……………………………………….34 圖六…………………………………………………………………………………………...35 圖七…………………………………………………………………….……………………..36 圖八………………………………………………………………….………………………..37 圖九…………………………………………………………………………………………...38 圖十…………………………………………………………………………………………...39 圖十一…………………………………………..…………………………………………….40 圖十二………………………………………………...………………………………………41 圖十三……………………………………..………………………………………………….42 圖十四……………………………………………………...…………………………………43 圖十五…………………………………………………………………………………….…..44 圖十六…………………………………...……………………………………………………...45 圖十七…………………………………………………………………………………………..46 圖十八…………………………………………………………………………………………..47 附錄一…………………………………………………………………………………………..48 附錄二…………………………………………………………………………………………...49

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