Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36104
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dc.contributor張邦彥zh_TW
dc.contributor陳浩仁zh_TW
dc.contributor.advisor孟孟孝zh_TW
dc.contributor.author李嘉芳zh_TW
dc.contributor.authorLi, Chia -Fangen_US
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T07:53:52Z-
dc.date.available2014-06-06T07:53:52Z-
dc.identifierU0005-1007200616253200zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/36104-
dc.description.abstract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zh_TW
dc.description.abstractAbstract 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.en_US
dc.description.tableofcontents目錄 中文摘要…………………………………………………………..…...…ⅰ 英文摘要…………………………………………………………..…...…ii 目錄………………………………………………………………..……...ⅲ 表目錄……………………………………………………………..…...…ⅴ 圖目錄……………………………………………………………..…...…vi 附錄目錄…..……………………………………………………......…...vii 第一章、 前言………………………………………………..…….………1 第二章、 材料與方法………………………………………………….…..8 第一節、 菌株、載體及培養基………………….………………..…..8 第二節、 質體的構築.………………………………………………..9 第三節、 大腸桿菌(E. coli)質體DNA之抽取…………………….15 第四節、 大腸桿菌之轉型作用(transformation)………..………...16 第五節、 SARS–CoV非結構性蛋白酵素之表達….………………17 第六節、 酵母菌之轉型作用(transformation)…………………….19 第七節、 SARS–CoV非結構性蛋白酵素以酵母菌之表達……….20 第八節、 蛋白質電泳分析法(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)………………………..20 第九節、 西方墨點法………………………………………..……...21 第十節、 利用細胞外轉錄及細胞外轉譯作用做非結構性蛋白質 的表達………………………………………..…………...22 第十一節、SARS-CoV非結構性蛋白質之戴帽酵素活性分析…....24 第三章、 結果…………………………………………………………….28 第一節、 以大腸桿菌表達非結構性蛋白質嘗試過的策略….…....28 (一)、直接利用含有非結構性蛋白質的質體在大腸桿菌表達 蛋白質……………………………………………………28 (二)、 與chaperone共表達…………………….……………….…28 (三)、將SARS-CoV非結構性蛋白質做共表達(co-expression 或以彼此融合的方式做蛋白質表達(fusion protein)….....29 第二節、 以酵母菌表達非結構性蛋白質與活性分析…….………29 (一)、以酵母菌表達非結構性蛋白質…………....……………...29 (二)、 nsp4戴帽酵素活性分析…………………………….........30 第三節、以Retic lysate表達非結構性蛋白質與活性分析…….......31 (ㄧ)、以Retic lysate表達SARS-CoV非結構性蛋白質..................31 (二)、戴帽酵素活性分析…………………………………….......33 第四章、 討論…………………………………………….………………33 第一節、 非結構性蛋白質的表達………………………………….35 第二節、 戴帽酵素活性測試…………..………………..………….35 參考文獻………………………………………………………………….38 表目錄 表一、pET32a–nsp2、pET32a–nsp3N、pET32a–nsp3C、pET32a–nsp4 分別在不同菌株表達的整理……………....................….............43 表二、利用不同的chaperone plasmids與pET32a–nsp2、pET32a– nsp3C、pET32a–nsp4在E. coli Origami B(DE3)表達的統理…..43 表三、共表達質體(pETDuet-1/nsp3N –nsp3C、pCDFDuet-1/nsp1–nsp 2) 分別在不同品系的大腸桿表達……………...………...…44 表四、pETDuet-1 /nsp1~nsp2(265~2718)、pETDuet-1/nsp2~nsp3N (8 02~3030)、pETDuet-1 /nsp2~nsp3N(802~3276)分別不同品系 的大腸桿菌表達……………………………………………...44 表五、由台灣大學所獲得的25個SARS Coronavirus (TW1) Clones…...45 表六、實驗中所有使用到的primers…………………………………......47 表七、實驗中所有使用到的chaperone plasmids………………………...50 表八、Kiwibrew的成分…………………………………………………..50 圖目錄 圖一、冠狀病毒非結構性蛋白質(nsp2、nsp3N、nsp3C、nsp4) 的表達。菌株為E. coli Rosetta(DE3)pLysS……………….......51 圖二、冠狀病毒非結構性蛋白質(nsp2、nsp3C、nsp4)的表達。 質體為pET32a,菌株為E. coli Rosetta-gamiTM(DE3)pLysS....52 圖三、利用不同的chaperone plasmid與目標蛋白質共同表達………....53 圖四、冠狀病毒非結構性蛋白質(nsp2、nsp3N、nsp3C)的表達。 質體為pYES2/CT ,菌株為S. cerevisiae INVSc1........................54 圖五、冠狀病毒非結構性蛋白質(nsp2、nsp3N、nsp3C)的表達。 質體為pYES2/NT ,菌株為S. cerevisiae INVSc1........................55 圖六、以in vitro transcription及Retic lysate的方法進行非結構性蛋 白質nsp2表達................................................................................56 圖七、以in vitro transcription及Retic lysate的方法進行非結構性蛋 白質nsp3C、 nsp4表達………………………………….……...57 圖八、In vitro translation BaMV 戴帽酵素活性測試...............................58 圖九、冠狀病毒非結構性蛋白質nsp2、 nsp3C 、 nsp4戴帽酵素 活性試………………………………………………...…………..59 圖十、利用薄層色層分析法(TLC)分析冠狀病毒非結構性蛋白質 nsp2、 nsp3C 、 nsp4是否有幫助GTP接到GDP上形成 GpppG的活性…………………………………………...………60 圖十一、SARS-CoV 5’UTR是否有IRES功能之分析……..…….……...61 附錄目錄 附錄一、電子顯微鏡下的SARS-CoV病毒顆粒………….….………….62 附錄二、SARS-CoV基因組功能與表現……………………..…….…….63 附錄三、SARS-CoV病毒顆粒的模型圖…………………………………64 附錄四、冠狀病毒在細胞內的生活史…………………………………...65 附錄五、SARS-CoV各個非結構性蛋白的功能……………………...….66 附錄六、質體p2luc之圖譜與特徵............…………………………….....67附錄七、pYES2-CT/nsp2構築的序列…….…………………………...…68附錄八、pYES2-CT/nsp3N構築的序列……………….……………...….69附錄九、pYES2-CT/nsp3C構築的序列……………………………...…..70 附錄十、pYES2-CT/nsp4構築的序列……………...………………...…..71 附錄十一、pYES2-NT/nsp2構築的序列……………...……………….....72 附錄十二、pYES2-NT/nsp3C構築的序列……………...………………..73 附錄十三、pYES2-NT/nsp4構築的序列……………...……………...…..74 附錄十四、pCDFDuet-1/sars-nsp1&nsp2構築的序列………………..…75 附錄十五、pETDuet-1/sars-nsp3N&nsp3C構築的序列...........................76 附錄十六、pETDuet-1/sars-nsp1~nsp2(265~2718)構築的序列……...….77 附錄十七、pETDuet-1/sars-nsp2~ nsp3N(820~3030)構築的序列…..…..78 附錄十八、pETDuet-1/sars-nsp2~nsp3N(820~3276)構築的序列…….....79 附錄十九、p2luc/ SARS 5’UTR構築的序列………………………….....80zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物科技學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1007200616253200en_US
dc.subjectSARS coronavirus capping enzyme activity assayen_US
dc.subject冠狀病毒之戴帽酵素活性分析zh_TW
dc.titleSARS冠狀病毒非結構性蛋白質-2、-3、-4 之表達及功能探討zh_TW
dc.titleProtein expression and activity examination of the nsp2, nsp3, and nsp4 of SARS Coronavirusen_US
dc.typeThesis and Dissertationzh_TW
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