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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 | Yi-Chieh Wu | en |
dc.contributor.author | 吳怡潔 | zh_TW |
dc.date.accessioned | 2021-05-20T22:00:02Z | - |
dc.date.available | 2012-09-09 | |
dc.date.available | 2021-05-20T22:00:02Z | - |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10801 | - |
dc.description.abstract | 登革病毒(Dengue virus,DENV)分類上屬於黃病毒科(Flaviviridae)中的黃病毒屬(Flavivirus)之成員,共有四種血清型(DENV1,DENV 2,DENV 3和DENV4),可造成熱帶及亞熱帶地區最重要的節肢動物傳播疾病。
登革病毒顆粒表面的外套膜蛋白質(Envelope protein,E),是決定病毒進入宿主細胞及膜融合的重要因子,並且也是宿主抗體辨識的主要目標。根據X線繞射晶體結構(X-ray crystallography)分析,其N端包含三個區域(domain),根據先前的研究報導,domain II之fusion loop為黃病毒群交叉反應(flavivirus group-reactive)抗體的主要辨識部位(epitopes),domain III包含複合型/次複合型交叉反應(complex/subcomplex-reactive)和血清型專一(type-specific)的抗體辨識部位。 由於過去的研究主要著重在感染DENV2後血清中抗E抗體的研究,對於感染其他血清型後之抗E抗體反應尚未瞭解。此外,大多數老鼠單株抗體之抗體辨識部位的數據主要來自DENV2 E蛋白質的研究。本研究第一個目標透過西方墨點法來研究DENV3 E蛋白質上其交叉反應及血清型專一的抗體辨識部位。以DENV3前驅膜蛋白質(Precursor membrane protein,PrM)及E蛋白質的表現質體為骨幹,在28個E蛋白質露出表面的胺基酸製造丙胺酸(alanine)定點突變,檢視老鼠單株抗體和DENV3病人血清多株抗體對於E蛋白質結合能力的影響。結果顯示domain II之fusion loop的某些胺基酸點突變會降低flavivirus group-reactive單株抗體結合能力,利用蛋白質結構為基礎的分析(structure-based modeling)顯示這些胺基酸之間的距離均符合同一個抗體辨識部位的範圍。domain III及domain I之某些胺基酸點突變會降低complex/subcomplex-reactive單株抗體結合能力,domain I之胺基酸點突變也影響所研究的一個type-specific單株抗體結合能力,蛋白質結構為基礎的分析顯示這些胺基酸符合形成相同的抗體辨識部位,E蛋白質上fusion loop和少數domain I胺基酸點突變則降低大多數感染DENV3病人血清抗E抗體的結合能力,顯示fusion loop是血清中主要抗E抗體的辨識部位。 本研究第二個目標,使用類病毒顆粒(virus-like particles,VLPs)建立capture ELISA系統來確認E蛋白質的抗體辨識部位,結果顯示fusion loop多點胺基酸突變VLPs會明顯降低感染DENV3病人血清抗E抗體的結合能力,利用終點稀釋倍數(endpoint titer)計算,顯示這群辨識fusion loop胺基酸(101W,108F)的抗E抗體所佔比例約為全部抗E抗體之20至70%。 綜合以上,本研究之結果顯示感染DENV的病人血清中主要抗E抗體辨識domain II fusion loop,另一方面,利用DNEV3 E蛋白質為骨幹的胺基酸點突變對不同的老鼠單株抗體結合能力的分析顯示,其辨識部位大致與之前文獻利用其他血清型E蛋白質所做的研究結果一致,但是也找到一些特殊的抗體辨識部位。這些結果提供對於交叉反應及血清型專一之抗體辨識部位的瞭解,以及對自然感染DENV3之下所誘發人體產生的抗E抗體特性的瞭解,可以對日後設計登革病毒重組疫苗提供有用的資訊。 | zh_TW |
dc.description.abstract | Dengue virus(DENV)belongs to the genus Flavivirus in the family Flaviviridae. The four serotypes of DENV(DENV1, DENV 2, DENV 3 and DENV4)are the leading cause of arboviral diseases in the tropical and subtropical areas.
The envelope(E)protein is known to play the critical role in virus entry and membrane fusion, as well as the major target of neutralizing antibodies. X-ray crystallographic analyses of the N-terminal ectodomain of E protein revealed three distinct domains. It has been shown that the fusion loop of domain II contains flavivirus group-reactive epitopes, whereas domain III contains complex/subcomplex-reactive and type-specific epitopes. Previous studies of anti-E antibodies in polyclonal human sera focused primarily on DENV2 patients and anti-E antibody responses after infection by other serotypes remains largely unknown. Moreover, the epitope mapping of most mouse monoclonal antibodies(mAbs)was based on DENV2 E protein. In the first specific aim of this study, we investigated the cross-reactive and type-specific epitopes on the E protein of DENV3 by Western blot. We generated 28 alanine-substitution mutants which were predicted as surface exposed E residues by site-directed mutagenesis on a DENV3 Precursor membrane(PrM)/E expression construct, and examined the E-binding activity of mouse mAbs and polyclonal human sera. Substitution of several fusion loop residues at domain II reduced the E-binding activity of flavivirus group-reactive mAbs, and structure-based modeling suggested that these fusion loop residues were within the range of an epitope. Substitution of some domain III or domain I residues reduced the E-binding activity of complex/subcomplex-reactive mAbs. Mutations of a few domain I residues also reduced the E-binding activity of one type-specific mAb studied. The E-binding activity of anti-E antibodies of most DENV3-infected patients’ sera were affected by mutations of fusion loop residues as well as domain I residue(two case), suggesting that fusion loop is the major epitope recognized by anti-E antibodies in human sera. In the second specific aim of this study, we established capture ELISA by using virus-like particles(VLPs)to confirm the epitopes identified by Western blot. Our results showed that VLPs containing multiple mutations of fusion loop residues reduced the E-binding activity of anti-E antibodies in DENV3-infected patients’ sera. Based on the endpoint titers of capture ELISA, the proportion of the anti-E antibodies recognizing the fusion loop residues(101W, 108F)ranged from 20% to 70%. In summary, our study suggests that anti-E antibodies in DENV patients’ sera primarily recognized fusion loop residues of domain II. Moreover, the epitopes of different mouse mAbs identified by amino acid substitutions in the DENV3 E protein backbone were generally in agreement with previous reports based on E protein of other serotypes. A new epitope recognized by a type-specific anti-E mAb was also found. The results presented in this study could further our understanding of the cross-reactive and type-specific epitopes, as well as the anti-E antibodies in polyclonal human sera. These information will be valuable for future design of recombinant subunit vaccine against DENV. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T22:00:02Z (GMT). No. of bitstreams: 1 ntu-99-R97445108-1.pdf: 2609840 bytes, checksum: eac92a537611dc3c0696083ec3c23ff1 (MD5) Previous issue date: 2010 | 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 第1章:序論 1 1.1 登革病毒之簡介及流行 1 1.2 感染登革病毒之臨床症狀 2 1.3 登革出血熱之致病機制 3 1.4 登革病毒之結構與基因體 4 1.5 登革病毒之前驅膜/膜蛋白質與外套膜蛋白質 4 1.6 登革病毒之生活史 5 1.7 類病毒顆粒 6 1.8 外套膜蛋白質之抗原結構 6 1.9 研究目的 8 第2章:材料與方法 10 2.1 質體的構築 10 2.2 菌種 11 2.3 質體轉形作用 12 2.4 製備小量質體 12 2.5 製備大量質體 13 2.6 細胞培養 13 2.7 細胞轉染試驗 13 2.8 西方墨點分析法 14 2.9 影像分析軟體 15 2.10 超高速離心分離重組次病毒顆粒 15 2.11 抗原捕捉酵素連結免疫分析法 16 2.12 病人血清 16 2.13 單株抗體 16 第3章:結果 18 3.1 老鼠單株抗體位於DENV3 E蛋白質之抗體辨識部位 18 3.2 感染DENV3病人血清抗E抗體之辨識部位分析 19 3.3 利用VLPs建立capture ELISA system研究DENV3 E蛋白質之 抗體辨識部位 20 3.4 分析病人血清中辨識fusion loop之抗E抗體所佔的比例 21 第4章:討論 22 第5章:參考文獻 26 圖表附錄 34 | |
dc.language.iso | zh-TW | |
dc.title | 探討單株抗體及人類血清位於第三型登革病毒外套膜蛋白質之抗體辨識部位 | zh_TW |
dc.title | Study of the Epitopes on the Envelope Protein of Dengue Virus Type 3 Recognized by Monoclonal Antibodies and Human Sera | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳漢忠,伍安怡,張淑媛 | |
dc.subject.keyword | 登革病毒,外套膜蛋白質,單株抗體,人類血清多株抗體,抗體辨識部位, | zh_TW |
dc.subject.keyword | dengue virus,envelope protein,monoclonal antibody,polyclonal human sera,epitope, | en |
dc.relation.page | 50 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-07-19 | |
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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