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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗(Shih-Chung Chang) | |
| dc.contributor.author | Jyun-Yu Ciou | en |
| dc.contributor.author | 邱俊瑜 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:36:20Z | - |
| dc.date.available | 2021-07-10T21:36:20Z | - |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76756 | - |
| dc.description.abstract | 2019年12月，新型冠狀病毒 (SARS-CoV-2) 最先在中國武漢的華南海鮮市場出現感染案例。現今已經造成了全球大流行，超過兩千一百萬感染案例及七十萬死亡案例。目前並無有效的抗體或藥物能夠治療 SARS-CoV-2的感染，也尚未研發出疫苗，因此能夠快速並精準地檢測SARS-CoV-2，是目前能夠延緩疫情蔓延的重要任務。 本論文利用大腸桿菌表現SARS-CoV-2的重組棘蛋白N-terminal domain (NTD)、receptor binding domain (RBD)、S1、S2、S全長與核衣殼蛋白(nucleocapsid protein; NP)；除NP重組蛋白為可溶外，以大腸桿菌表現的NTD、RBD、S1、S2與S重組蛋白皆存在於inclusion body中，因此利用含8 M尿素的溶液使蛋白質溶解，再以HisTrap層析管柱進行純化，得到高純度的重組蛋白。接著利用劃線機將重組蛋白塗佈到PVDF膜上，再與血清樣本反應後，以西方墨點法偵測訊號。實驗結果顯示確診Coronavirus disease 2019 (COVID-19) 之患者的血清結合於NP重組蛋白之訊號為最強，但是正常人之血清樣本亦有些許的交叉反應；S2、S與NTD重組蛋白的訊號較弱，但是專一性較高，正常人之血清樣本並無交叉反應；使用RBD重組蛋白為抗原時並無偵測到任何訊號；而使用S1重組蛋白為抗原的專一性不佳，確診者與正常人之血清樣本皆可獲得訊號，無法正確診斷是否受到SARS-CoV-2的感染。本論文研究結果顯示，使用大腸桿菌表現之NP、NTD、S2與S重組蛋白所製成的試片可運用於COVID-19的疾病診斷。 | zh_TW |
| dc.description.abstract | At the end of December, 2019, the new coronavirus (SARS-CoV-2) appeared in the seafood market in Wuhan, China. Recently, more than 21 million infection cases and 7 hundred thousands of death cases have been reported. Currently, there is no effective antibody or drug that can treat SARS-CoV-2 infection, and no vaccine has yet been successfully developed. Therefore, the ability to detect SARS-CoV-2 quickly and accurately is an important task to prevent the spread of the epidemic. The research utilized E. coli to express the recombinant N-terminal domain (NTD), receptor binding domain (RBD) of Spike protein (S), S1, S2, S and nucleocapsid protein (NP) of SARS-CoV-2. Except NP, the NTD, RBD, S1, S2, S recombinant protein were expressed as inclusion body in E. coli. Therefore, a solution containing 8M urea was used to dissolve the protein, and the NTD, RBD, S1, S2, S and NP recombinant protein were purified with HisTrap chromatography column to obtain high purity of proteins. The recombinant proteins were coated on the PVDF membrane, and then incubated with the patient's serum for Western blotting analysis. The experimental results showed that the patient’s serum diagnosed as Coronavirus disease 2019 (COVID-19) positive has the strongest signal for binding to NP recombinant protein, but the normal patient’s serum also has a slight cross reaction with NP recombinant protein. The signals were weak by using S2, S and NTD recombinant protein as the antigens, but they were more specific for not causing any cross reaction. There was no signal while using RBD recombinant protein as the antigen. The specificity was not good while using S1 recombinant protein as the antigen since both of the COVID-19 positive and negative sera could obtain the signal. This research showed that the test strips coated with the E. coli-expressed NP, NTD, S2 and S recombinant protein can be applied for the diagnosis of COVID-19. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:36:20Z (GMT). No. of bitstreams: 1 U0001-1808202016380500.pdf: 2109522 bytes, checksum: c036c7e7aa1afbf6e0490cfa0c838e3c (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 目錄 I 摘要 V Abstract VI 縮寫表 VIII 第一章 緒論 1 1.1 新型冠狀病毒簡介 1 1.1.1 冠狀病毒 (Coronavirus) 1 1.1.2 新型冠狀病毒的基因體 2 1.1.3 新型冠狀病毒的蛋白質體 2 1.1.4 冠狀病毒的生活史及複製週期 5 1.2 乙型冠狀病毒 6 SARS-CoV與SARS-CoV-2的比較 7 1.3 SARS-CoV-2鑑定與檢測方法 7 1.3.1 即時反轉錄核酸檢測 8 1.3.2 血清學檢測 8 1.4 研究動機與目的 11 第二章 材料與方法 12 2.1 實驗材料 12 2.1.1 大腸桿菌DH5α 12 2.1.2 大腸桿菌BL21(DE3) 12 2.1.3 大腸桿菌Rosetta(DE3) 13 2.1.4 COVID-19陽性血清 13 2.2 表現載體建構 13 2.2.1 原核表現系統載體 13 2.2.2 核酸引子設計 13 2.2.3 聚合酶鏈鎖反應 (Polymerase chain reaction) 14 2.2.4 限制酶切反應 14 2.2.5 DNA接合反應 15 2.2.7 重組載體之篩選 15 2.3 核酸實驗方法 16 2.3.1 洋菜膠體電泳 16 2.3.2 核酸純化 16 2.3.3 核酸定量 17 2.3.4 質體DNA製備 17 2.4 蛋白質實驗方法 17 2.4.1 蛋白質定量 17 2.4.2 聚丙烯醯胺凝膠電泳 18 2.4.3 CBR染色法 18 2.4.4 蛋白質轉印法 19 2.4.5 西方墨點法 19 2.5 重組蛋白質表現 20 2.5.1 大腸桿菌株Rosetta(DE3)表現 20 2.5.2 大腸桿菌BL21(DE3)表現 20 2.6 重組蛋白純化 20 2.6.1 純化SARS-CoV-2 NTD, RBD, S1重組蛋白 20 2.6.2 純化SARS-CoV-2 S2, Spike重組蛋白 21 2.6.3 純化SARS-CoV-2 NP重組蛋白 22 2.6.4 蛋白質濃縮 23 2.6.5 蛋白質上樣劃線法 23 第三章 結果 25 3.1 大腸桿菌表現質體建構 25 3.1.1 NTD、RBD、S1、S2和S之表現載體建構 25 3.1.2 NP之表現載體建構 25 3.2 製備大腸桿菌表現重組蛋白並純化 26 3.2.1 SARS-CoV-2 NTD、RBD和S1重組蛋白於大腸桿菌之表現 26 3.2.2 SARS-CoV-2 S2, Spike於大腸桿菌之表現 27 3.2.3 SARS-CoV-2 NP於大腸桿菌之表現 27 3.3 SARS-CoV-2重組蛋白與COVID-19陽性血清結合測試 28 3.3.1 測定COVID-19患者血清內抗體對SARS-CoV-2重組蛋白之專一性 28 3.3.2 測定COVID-19患者血清內對抗SARS-CoV-2重組蛋白之IgG與IgM抗體之表現 28 第四章 討論 30 4.1 SARS-CoV-2重組蛋白表現與純化之探討 30 4.2 COVID-19檢測方法之探討 30 4.3 COVID-19陽性血清與SARS-CoV-2重組蛋白結合專一性之探討 31 4.4 COVID-19陽性血清中IgG和IgM抗體表現與COVID-19病程探討 32 4.5 COVID-19檢測平台應用之探討 32 參考文獻 34 圖與表 38 圖一 SARS-CoV-2棘蛋白示意圖 39 圖二 NTD、RBD、S1、S2和S表現載體之建構 40 圖三 NP表現載體之建構 41 圖四 以E.coli Rosetta(DE3) 表現NTD、RBD和S1 42 圖五 以E.coli Rosetta(DE3) 表現S2和Spike 44 圖六 以E.coli BL21(DE3) 表現NP 45 圖七 測定COVID-19患者血清內抗體對SARS-CoV-2重組蛋白之專一性 46 圖八 測定COVID-19患者血清內IgG與IgM之含量 47 圖九 SARS-CoV-2重組蛋白與COVID-19病患血清之結合訊號與專一性 48 附錄 49 | |
| dc.language.iso | zh-TW | |
| dc.subject | 檢測試片 | zh_TW |
| dc.subject | 新型冠狀病毒 | zh_TW |
| dc.subject | 棘蛋白 | zh_TW |
| dc.subject | 核衣殼蛋白 | zh_TW |
| dc.subject | 重組蛋白 | zh_TW |
| dc.subject | New coronavirus (SARS-CoV-2) | en |
| dc.subject | test strip | en |
| dc.subject | recombinant protein | en |
| dc.subject | Nucleocapsid protein | en |
| dc.subject | Spike protein | en |
| dc.title | 以大腸桿菌表現系統製備SARS-CoV-2重組棘蛋白和核衣殼蛋白 | zh_TW |
| dc.title | Production of the recombinant SARS-CoV-2 spike and nucleocapsid proteins by using the Escherichia coli expression system | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林翰佳(Han-Jia Lin),陳威戎(Wei-Jung Chen),廖憶純(Yi-Chun Liao) | |
| dc.subject.keyword | 新型冠狀病毒,棘蛋白,核衣殼蛋白,重組蛋白,檢測試片, | zh_TW |
| dc.subject.keyword | New coronavirus (SARS-CoV-2),Spike protein,Nucleocapsid protein,recombinant protein,test strip, | en |
| dc.relation.page | 51 | |
| dc.identifier.doi | 10.6342/NTU202003996 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-20 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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