Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36104
標題: SARS冠狀病毒非結構性蛋白質-2、-3、-4 之表達及功能探討
Protein expression and activity examination of the nsp2, nsp3, and nsp4 of SARS Coronavirus
作者: 李嘉芳
Li, Chia -Fang
關鍵字: SARS coronavirus capping enzyme activity assay;冠狀病毒之戴帽酵素活性分析
出版社: 生物科技學研究所
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摘要: 
中文摘要
SARS冠狀病毒,為單一正股RNA病毒,5’端具戴帽結構(cap),3’端為poly(A)-tail,基因體含約3萬個鹼基;由核酸序列推測其基因體含14個蛋白質轉譯架構(ORF)。轉譯架構1 (ORF1)負責生成一含4382與7073個胺基酸的蛋白質(分別為ORF1a與ORF1ab polyproteins),參考已知它種冠狀病毒研究結果,推測此巨大蛋白質會自行切解成16個非結構性蛋白質(non-structure protein,nsp),由於冠狀病毒繁衍於寄主細胞之細胞質中,但寄主細胞自身負責催化基因體5’端cap結構形成的酵素卻位於細胞核中,因此冠狀病毒應備有自己獨特的酵素活性,用以催化病毒基因體5’端cap結構的形成,而相關的活性很可能存在於這些非結構性蛋白中。因此,本實驗意圖探討SARS-CoV 的 nsp2、nsp3C和nsp4是否與病毒形成cap結構有關。首先,利用大腸桿菌表達蛋白質的策略,期望能得到蛋白質以分析其具有何種特性。可惜在大腸桿菌中以個別表達不同段的非結構性蛋白質的方式去生產蛋白質,卻無法成功,因此嘗試藉由其它方法幫助蛋白質表達如:與chaperonins共同表達、將不同區段非結構性蛋白質作共同表達(co-expression)、或將不同區段非結構性蛋白質做融合(fusion)成一段蛋白質,希望藉由此方式能順利表達出蛋白質,但似乎均無法得到期待效果。因此決定改用內質網紅血球細胞萃取液(retic lysate)來表達蛋白質。目前此方法已可偵測到病毒蛋白質,故實驗中採取此方法表達非結構性蛋白質並進行戴帽酵素的活性分析。目前雖嘗試應用不同方法去測試蛋白質是否有戴帽酵素的活性存在,但尚未發現這些非結構性蛋白質有戴帽酵素的活性存在,未來將改善活性測試的方法或者嘗試去測試其它非結構性蛋白質的活性,去確認在SARS-CoV的非結構性蛋白質是否有與病毒基因體5’端cap結構形成

Abstract
The single-stranded plus sense RNA genome of SARS coronavirus (SARS-CoV)has ~30,000 nucleotides with a 5' cap structure and 3' poly(A) tract, and contains 14 putative open reading frames in a similar organization to that of other coronavirus. The product of ORF1 (orf1ab) is a polyprotein consisting of 7073 amino acids that is predicted to be self cleaved, by two viral proteinases, into 16 nonstructural proteins. Because coronavirus replicates in the cytoplasm of the infected cells, it should be equipped with its own RNA capping apparatus. It is therefore reasonable to assume that some of the nonstructural proteins, derived from the cleavage of orf1ab polyprotein, harbor such respective enzymatic activities. In this experiment, we tried to overexpress some selected nonstructural proteins such as nsp2, nsp3C, and nsp4 in Escherichia coli. Many methods including coexpressing the nonstructural proteins with chaperonins, coexpressing more than two of the nonstructural proteins, or fusing the nonstructural protein together have been used. But it seems that Escherichia coli system can not express nonstructural proteins, nsp2, nsp3C, and nsp4. Therefore we try to use recticulocyte lysate for the in vitro translation of SARS-CoV nonstructural proteins. Several methods have been used to investigate whether the in vitro expressed nonstructural proteins have capping enzyme activity or not. However, no activity has been identified yet. In the future we will try to improve the methods for activity assay or try to detect the activities from other nonstructural proteins to confirm if these SARS-CoV nonstructural proteins are involved in the formation of 5'cap structure of the genomic RNA.
URI: http://hdl.handle.net/11455/36104
其他識別: U0005-1007200616253200
Appears in Collections:生物科技學研究所

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