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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王維恭(Wei-Kung Wang) | |
dc.contributor.author | I-Jung Liu | en |
dc.contributor.author | 劉薏蓉 | zh_TW |
dc.date.accessioned | 2021-06-12T18:04:10Z | - |
dc.date.available | 2018-12-31 | |
dc.date.copyright | 2008-02-19 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-01-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27414 | - |
dc.description.abstract | 嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome, SARS)起源於中國且於2003年間爆發疫情,造成世界近30多國的流行。其致病源為一種新型冠狀病毒,稱為SARS冠狀病毒(SARS coronavirus)。由於SARS本身具有相當高的傳染力,由實驗室快速且正確地偵測SARS冠狀病毒之存在,對於病人的早期診斷與治療及疫情的控制非常重要。
本研究的第一個目標是利用間接免疫螢光法,以嚴重急性呼吸道症候群病人漱口水(throat wash)中的上皮細胞及針對SARS冠狀病毒核殼蛋白質(nucleocapsid protein)之兔血清,發展出偵測SARS冠狀病毒之抗原偵測法。實驗結果顯示,在我們偵測的17個SARS病人中有11位呈現陽性反應(陽性率67%),最早可在發燒第二天偵測到,然而在10個健康的控制組中皆呈陰性反應。與SARS冠狀病毒的其他偵測法作比較,此方法簡便、快速且符合經濟效益。在未來如果SARS再度流行時,此偵測法可作為另一個可行的早期快速診斷的方法,特別是在無法進行SARS冠狀病毒培養或其反轉錄聚合酶鏈反應(RT-PCR)偵測的地區。 由於我們在SARS病人漱口水的研究中觀察到大量的黏液(mucus)。黏液的主要成分之一(mucin)是一種高度醣化的蛋白質,而SARS冠狀病毒之棘蛋白質(Spike protein, S)也是一種高度醣化的蛋白質,因此我們假設S蛋白質與黏液蛋白質有交互作用,可能與SARS冠狀病毒感染呼吸道且經由呼吸道飛沫(droplet)傳染有關。本研究的第二個目標是利用共同免疫沉澱法測試S蛋白質與呼吸道中黏液蛋白質之間的交互作用。我們利用由呼吸道中主要黏液蛋白質mucin 5AC的第4區域(domain)而來的NP3a基因片段所表現之黏液醣蛋白質與S蛋白質做共同免疫沉澱法並以西方墨點法測試。實驗結果顯示目前我們尚無法證實S蛋白質與黏液蛋白質mucin 5AC之間的交互作用。 SARS疫情雖已結束,可能再出現仍值得關注。由於目前尚無有效且特異的藥物,尋找SARS冠狀病毒抑制劑的標的以進一步研發藥物仍是當務之急。在SARS冠狀病毒的複製過程各步驟中,病毒進入細胞是一個具有潛力發展抑制劑的步驟。本研究的第三個目標是建立快速簡便的細胞融合試驗(cell fusion assay),並利用此方法探討SARS冠狀病毒病毒進入細胞的抑制劑。我們建立了兩種細胞融合試驗。第一種是在顯微鏡下直接觀察融合細胞並計數。第二種則是以β-galactosidase 為報告基因的SARS冠狀病毒細胞融合試驗。結果顯示,依據S蛋白質第二次單元S2的HR2(heptad repeat 2) 區域所設計的P1、P4、及P6胜肽鏈,在續列濃度稀釋下,具有很好之細胞融合抑制效果。其中P6為23mer,是目前已知最短的抑制胜肽鏈,其抑制濃度IC50為1.03μM。根據X光結晶繞射實驗得知,HR2之中心位置(第1160至1177個胺基酸)可形成一種5-turnα-helix,並與HR1(heptad repeat 1)區域的深谷區(deep groove)(第909-928個胺基酸)具有交互作用,此區被預測可作為SARS冠狀病毒進入細胞抑制劑的標的。P6由第1153至1175個胺基酸包括深谷區,支持X光結晶繞射實驗之預測。另外,我們設計HR1區域的N46胜肽鏈及N46eg突變胜肽鏈,結果顯示其抑制濃度IC50分別為3.82μM及5.07μM。以1:1比例混合N46及N46eg具有協同抑制作用,IC50為1.39μM。此為繼第一型人類免疫不全病毒之後,結合HR1胜肽鏈及突變的HR1胜肽鏈可達到協同抑制作用的第二個例子,顯示此種策略似乎可做為快速發展對抗其他第一型外套膜(class I enveloped virus)病毒進入細胞抑制劑的方式之一。 | zh_TW |
dc.description.abstract | Severe acute respiratory syndrome (SARS)originated in China and has spread to near 20 countries in 2003. The etiological agent is a novel coronavirus, the SARS associated CoV (SARS-CoV). Because of the relatively high transmissibility of SARS, rapid and accurate detection of SARS-CoV in the laboratory is important for the early diagnosis and treatment for patients as well as for the control of the disease.
The first aim of this study, is to establish an indirect immunofluorescence test, which utilized cells derived from throat wash samples of patients with SARS and a rabbit serum that recognized the nucleocapsid protein of SARS-CoV, as an antigen assay for SARS-CoV. It can detect SARS-CoV in 11 out of 17(65%)samples from SARS patients as early as day 2 of illness but in none of the 10 samples from healthy controls. Compared with other diagnostic methods for detecting SARS-CoV, this assay is simpler, more convenient, and economical. Should SARS return in the future, this assay can be an alternative for early and rapid diagnosis of SARS, especially in countries where virus isolation or RT-PCR is not available. From our study of throat wash samples from SARS patients, we found large amounts of mucus in throat wash. Since mucin, one of the major component of mucus and the spike(S)protein of SARS-CoV are heavily glycosylated glycoprotein, we hypothesized that S protein can interact with mucin and this interaction may contribute to the transmission of SARS-CoV through respiratory droplets. The second aim of this study is to examine the interactions between S protein and mucin by co-immunoprcipitation and Western blot analysis. Our results revealed no interaction between the domain 4 of mucin 5AC, one of the respiratory mucin, and S protein. Despite the SARS epidemic is over, concerns regarding the possibility of resurgence in the future remain. Since no specific and effective drug against SARS-CoV is available now, identification of potential targets of inhibitors for future drug development is important. Among the different steps of replication of SARS-CoV, viral entry is an attractive step for the development of inhibitors. The third aim of this study is to establish a rapid and convenient cell fusion assay to study the entry inhibitors of SARS-CoV. We have established two cell fusion assay; one relied on counting the number of syncytia under light microscope, and the other one utilied the β-galatosidase gene as reporter gene. We found three peptides, P1, P4 and P6, derived from the heptad repeat 2(HR2)at the S2 subunit of S protein had good inhibitory effects. P6, a 23-mer peptide, is the smallest inhibitory peptide with an IC50 of 1.03 μM. Based on the X-ray crystallographic studies of core structures of S protein, a 5-turn α-helix at the central region of HR2(residues 1160 to 1177) packed against a relatively deep groove(residues 909 to 928)of HR1, which was predicted to be a potential target for entry inhibitors of SARS-CoV. The identification of P6(residues 1153 to 1175), which interacted with the deep groove of HR1, supported the prediction from X-ray crystallography. The HR1 peptide, N46, and its mutated version, N46eg, can inhibit the fusion with the IC50 of 3.82μM and 5.07μM, respectively. Moreover, combination of N46 and N46eg showed synergistic inhibition with an IC50 of 1.39μM. Our findings of synergistic inhibition between an HR1 peptide and a mutated HR1 peptide, a second such observation for class I enveloped viruses after human immunodeficiency virus type 1, suggested that this can be a strategy for quick development of entry inhibitors for other class I envelope viruses. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:04:10Z (GMT). No. of bitstreams: 1 ntu-97-D90445001-1.pdf: 3470054 bytes, checksum: 1a64b35ba590215a1ad14d4b533e4a0f (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 口試委員會審定書………………………………………………………I
誌謝………………………………………………………………………II 中文摘要………………………………………………………………III 英文摘要…………………………………………………………………V 壹、緒論…………………………………………………………………1 一、SARS冠狀病毒之起源及流行病史…………………………………1 二、SARS冠狀病毒之分類、結構及基因體……………………………2 三、SARS冠狀病毒之複製過程…………………………………………5 四、SARS冠狀病毒之感染及其臨床表現………………………………6 五、SARS冠狀病毒之偵測………………………………………………7 六、SARS冠狀病毒之治療………………………………………………9 七、黏液蛋白質…………………………………………………………11 貳、研究目的……………………………………………………………13 參、材料與方法…………………………………………………………14 一、檢體來源及製備……………………………………………………14 二、間接免疫螢光法 (Indirect immunofluorescence assay, IFA) ……………………………………………………………………………14 三、質體的構築 (Plasmid construction) …………………………15 四、菌種 (Bacteria strain) ………………………………………21 五、製備勝任細胞(Competent cell)………………………………21 六、轉形作用(Transformation)……………………………………21 七、製備小量質體 (Mini-plasmid preparation) …………………22 八、製備大量質體 (Maxi-plasmid preparation) …………………22 九、轉染作用 (Transfection)……………………………………22 十、病毒株………………………………………………………………23 十一、細胞培養 (Cell culture)…………………………………23 十二、細胞融合抑制試驗 (Cell fusion inhibition assay) …………………………………………………………………………24 十三、西方墨點法 (Western blot) …………………………………25 十四、抗體………………………………………………………………26 十五、病毒核酸之萃取…………………………………………………26 十六、即時核酸定量系統定量病毒RNA………………………………27 十七、胜肽鏈之合成…………………………………………………28 十八、MTT 細胞毒性試驗………………………………………………28 十九、Circular dichroism (CD)光譜之分析………………………28 二十、共同免疫沉澱(Immunoprecipitation)……………………28 肆、結果…………………………………………………………………31 伍、討論…………………………………………………………………39 陸、參考文獻……………………………………………………………44 圖表目錄 表一、利用間接免疫螢光法偵測SARS病人及健康者漱口水中上皮細胞中的SARS冠狀病毒………………………………………………………60 表二、以細胞融合試驗測試來自國家衛生研究院之化合物…………61 表三、摘要SARS冠狀病毒S蛋白質中HR1及HR2區域所衍生之抑制性胜肽鏈………………………………………………………………………62 圖一、利用間接免疫螢光法偵測 SARS病人及健康者漱口水中上皮細胞中的SARS冠狀病毒……………………………………………………63 圖二、構築質體 pCMV-NP3a1 及pCMV-NP3a2 之示意圖……………64 圖三、構築質體 12kpn-S1 、12NHA1-S1、12NHA2-S1、12NHA3-S1及 12CHA-S1之示意圖及其表現……………………………………………65 圖四、以共同免疫沈澱法偵測S1蛋白質與黏液蛋白質之交互作用…66 圖五、構築質體 pcDNA-HA-ACE2及S(h)之示意圖及其表現…………67 圖六、細胞融合試驗……………………………………………………68 圖七、以細胞融合試驗測試一批工業技術研究院之化合物…………69 圖八、以細胞融合試驗測試一批來自國家衛生研究院之化合物……70 圖九、以β-galactosidase 為報告基因的細胞融合試驗…………71 圖十、設計SARS 冠狀病毒之S 蛋白質抑制性胜肽鏈示意圖………72 圖十一、HR2 胜肽鏈之細胞融合抑制作用……………………………73 圖十二、HR1 胜肽鏈之細胞融合抑制作用……………………………74 圖十三、MTT 細胞毒性試驗……………………………………………75 圖十四、Circular dichroism (CD)光譜測試胜肽鏈之二級結構…76 | |
dc.language.iso | zh-TW | |
dc.title | 嚴重急性呼吸道症候群冠狀病毒的抗原偵測及其進入細胞抑制劑之研究 | zh_TW |
dc.title | Antigen Detection and Study of Entry Inhibitors of the Severe Acute Respiratory Syndrome Associated Coronavirus | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 張明富,高全良,董馨蓮,葉秀慧 | |
dc.subject.keyword | 嚴重急性呼吸道症候群,間接免疫螢光法,棘蛋白質,細胞融合試驗, | zh_TW |
dc.subject.keyword | Severe acute respiratory syndrome,indirect immunofluorescence test,Spike protein,cell fusion assay, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-01-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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