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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡錦華 | zh_TW |
| dc.contributor.advisor | Ching-Hwa Tsai | en |
| dc.contributor.author | 林豔玲 | zh_TW |
| dc.contributor.author | Yan-Ling Lin | en |
| dc.date.accessioned | 2023-09-05T16:07:08Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-05 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89209 | - |
| dc.description.abstract | 登革熱 (dengue fever) 是由登革熱病毒 (dengue virus, DENV) 引起的一種急性傳染病，主要透過病媒蚊蟲傳播給人類。該疾病在全球範圍內廣發流行，特別是在熱帶和亞熱帶地區，對公共衛生造成重大挑戰。登革熱感染由四種不同血清型的登革熱病毒引起，由於目前缺乏安全有效的疫苗和藥物，早期診斷可以提高患者的治療效果和生存率。登革熱病毒的非結構蛋白NS1，是一種高度免疫原性的醣蛋白，可以由登革熱感染的宿主細胞持續分泌，被認爲是一個潛在的診斷生物標誌物。然而，市售的NS1抗原檢測靈敏度有限，並且容易與其他黃病毒的NS1蛋白發生交叉反應。因此，生產製備高靈敏度的抗NS1蛋白抗體對於開發NS1蛋白快速檢測方法至關重要。
本研究的目標是篩選和鑑定針對登革熱病毒NS1蛋白的專一性單株抗體，以提高未來登革熱的臨床早期診斷和血清型鑑定的準確性。本研究先對四種血清型登革熱病毒的多種毒株進行親緣關係樹分析，首次確認了NS1蛋白具有分型登革熱病毒的能力。我們基於實驗室現有的114個抗NS1蛋白之單株抗體，經由西方墨點法和斑點印跡法初步篩選，確認了114個單株抗體的登革熱病毒血清型特異性。其中僅有1個單株抗體靶向DENV1-4 NS1蛋白的保守且線性區域，8個單株抗體具有與登革熱病毒和茲卡病毒 NS1蛋白高度保守區域 (conserved region) 的抗原決定位 (epitopes)，6個單株抗體具有DENV-2 strain 16681 NS1線性抗原決定位，還有10個單株抗體爲DENV-3 NS1線性抗原決定位。 此外，本研究還利用大腸桿菌BL21細胞表現NS1重組蛋白，以確認篩選出的單株抗體是否能夠辨識近年流行之登革熱病毒NS1蛋白，亦可作爲免疫小鼠的免疫源製備更多的單株抗體。然而在細菌表現系統中，NS1重組蛋白容易存在於不可溶的包涵體，需要進行變性處理才能增加其可溶性。變性處理可能導致蛋白失去天然的結構和功能，使本研究無法進一步確認單株抗體是否能夠識別天然形式NS1蛋白。經過登革熱病毒血清型2 NS1重組蛋白免疫兩次的小鼠血清中，抗登革熱病毒血清型2 NS1蛋白的抗體效力佳，卻也存在對其他DENV血清型及茲卡病毒NS1蛋白有交叉反應的抗體。 最後，我們評估了具有登革熱病毒NS1蛋白表面連續抗原決定位的單株抗體對其他黃病毒NS1蛋白的交叉反應。我們發現其中15個單株抗體與日本腦炎病毒NS1蛋白存在交叉反應。進一步的實驗揭示了這些單株抗體對日本腦炎病毒NS1蛋白的抗原決定位類型，其中有部分單株抗體具有構象抗原決定位，而其他能夠識別日本腦炎病毒變性的NS1/NS1’蛋白的單株抗體，展示出其對登革熱病毒NS1蛋白高度保守的線性抗原決定位識別的能力。 總而言之，本研究的成果對於提高登革熱的早期檢測和分型能力具有重要意義。然而，仍需要進一步的研究來改進抗原的免疫原性、提高抗體的專一性，並在臨床實踐中驗證其效能. | zh_TW |
| dc.description.abstract | Dengue fever is an acute infectious disease caused by dengue virus (DENV), primarily transmitted to humans through vector mosquitoes. This disease has been widely prevalent globally, particularly in tropical and subtropical regions, posing significant challenges to public health. Dengue is caused by four different serotypes of dengue virus. Early diagnosis is essential to improve patient treatment outcomes and survival rates due to the lack of safe and effective vaccines and drugs. The non-structural protein 1 (NS1) of dengue virus is a highly immunogenic glycoprotein and is considered as a potential diagnostic biomarker since it is continuously secreted by host cells during dengue infections. However, commercially available NS1 antigen tests have limited sensitivity and are prone to cross-reactivity with NS1 proteins of other flaviviruses. Therefore, it is crucial to produce highly specific anti-NS1 antibodies in order to develop a rapid test for NS1 detection.
The objective of this study is to clarify and characterize monoclonal antibodies specifically targeting the NS1 protein of dengue virus, aiming to enhance the accuracy of clinical early diagnosis and serotyping of dengue viruses. Phylogenetic tree analysis was performed on multiple strains of the four dengue virus serotypes, confirming for the first time that the NS1 protein has serotyping capabilities. Based on our existing panel of 114 monoclonal antibodies against NS1 protein, we conducted preliminary characterization using western blot and dot blot assays, confirming the serotype specificity of the 114 monoclonal antibodies. Among them, only one monoclonal antibody targets the conserved and linear region of DENV1-4 NS1 protein, eight monoclonal antibodies recognize highly conserved epitope in the NS1 proteins of both dengue and Zika viruses, six monoclonal antibodies target linear antigenic determinants of DENV-2 strain 16681 NS1, and ten monoclonal antibodies only target linear antigenic determinants of DENV-3 NS1. Additionally, we expressed recombinant NS1 protein in Escherichia coli BL21 cells to verify whether the selected monoclonal antibodies can recognize the NS1 protein of recently circulating DENV and to produce more monoclonal antibodies as immunogens for mice. However, in the bacterial expression system, recombinant NS1 protein tends to form insoluble inclusion bodies, requiring denaturation treatments to enhance solubility. Urea treatments may lead to the loss of native conformation and function of recombinant NS1 protein, which hinders the subsequent validation of monoclonal antibody recognition towards the native NS1 protein. Antibodies against DENV-2 NS1 protein showed good efficacy in mouse sera after immunization with recombinant NS1 protein twice. However, they still exhibited cross-reactivity with other DENV serotypes and NS1 protein of the Zika virus. Finally, we evaluated the cross-reactivity of monoclonal antibodies targeting linear epitopes on the surface of dengue virus NS1 protein with NS1 proteins from other flaviviruses. We found that 15 monoclonal antibodies exhibited cross-reactivity with Japanese encephalitis virus NS1 protein. Further experiments revealed the antigenic determinant types of these monoclonal antibodies. Some of the monoclonal antibodies exhibited conformational epitopes, while others demonstrated the ability to recognize linear epitopes on denatured NS1/NS1' proteins of Japanese encephalitis virus. These findings showcased their highly conserved linear epitope recognition ability for the NS1 protein of the dengue virus. In conclusion, the findings of this study hold significant implications for improving early detection and serotyping capabilities for dengue virus. However, further research is needed to enhance the immunogenicity of antigens, improve antibody specificity, and validate their efficacy in clinical practice. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-05T16:07:08Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-05T16:07:08Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 中文摘要 I
Abstract III 第一章 序論 1 1.1. 登革熱病毒 1 1.2. 登革熱病毒非結構性蛋白NS1 6 1.3. 單株抗體 (monoclonal antibody) 12 1.4. 研究目的 15 第二章 材料與方法 16 2.1. 實驗材料 16 2.2. 實驗方法 23 第三章 結果 32 3.1. 抗NS1蛋白單株抗體的登革熱病毒血清型鑑定 32 3.2. 探討南美洲和亞洲七年內登革熱病毒 NS1蛋白突變情形 35 3.3. 製備單株抗體所用的登革熱病毒NS1抗原蛋白質 36 3.4. 小鼠免疫反應 38 3.5. 抗登革熱病毒NS1蛋白之單株抗體的交叉反應 39 第四章 討論 43 第五章 圖表 52 表一、實驗中所使用之引子列表 52 表二、實驗中所使用之抗體列表 53 圖一、登革熱病毒基因組轉譯蛋白示意圖 54 圖二、登革熱病毒之生活史 55 圖三、亞洲七國登革熱病例總數分佈地理圖 56 圖四、登革熱病毒NS1蛋白結構示意圖 57 圖五、登革熱病毒NS1蛋白參與補體拮抗之示意圖 58 圖六、pET21a(+)-DENV1-NS1質體示意圖 59 圖七、pET21a(+)-DENV2-NS1質體示意圖 60 圖八、pET21a(+)-DENV3-NS1質體示意圖 61 圖九、pET21a(+)-DENV4-NS1質體示意圖 62 圖十、利用西方墨點法和斑點印跡法探查抗DENV-1 NS1蛋白的單株抗體之專一性 63 圖十一、利用西方墨點法和斑點印跡法探查抗DENV-2 16681 strain NS1蛋白的單株抗體之專一性 65 圖十二、利用西方墨點法探查抗DENV-3 NS1蛋白的單株抗體之專一性 66 圖十三、利用斑點印跡法探查抗DENV-3 NS1蛋白的單株抗體之專一性 67 圖十四、利用西方墨點法探查抗DENV-4 NS1蛋白的單株抗體之專一性 68 圖十五、利用西方墨點法和斑點印跡法探查抗登革熱病毒四種血清型NS1蛋白的單株抗體之特異性 69 圖十六、利用西方墨點法探查抗登革熱病毒和茲卡病毒NS1蛋白的單株抗體之特異性 70 圖十七、利用斑點印跡法探查抗登革熱病毒和茲卡病毒NS1蛋白的單株抗體之特異性 71 圖十八、利用斑點印跡法探查114個單株抗體之登革熱病毒血清型特異性 73 圖十九、利用西方墨點法探查114個單株抗體之登革熱病毒血清型特異性 75 圖二十、七年內南美洲和亞洲DENV-1 NS1胺基酸（81-160 a.a.）之序列比對 76 圖二十一、七年內南美洲和亞洲DENV-2 NS1胺基酸（161-240 a.a.）之序列比對 77 圖二十二、七年內南美洲和亞洲DENV-3 NS1胺基酸（161-240 a.a.）之序列比對 78 圖二十三、七年內南美洲和亞洲DENV-4 NS1胺基酸（81-160 a.a.）之序列比對 79 圖二十四、七年內南美洲和亞洲四種血清型登革熱病毒NS1胺基酸親緣關係樹 80 圖二十五、四種血清型登革熱病毒NS1胺基酸之序列比對 81 圖二十六、利用聚合酶連鎖反應挑選含目標質體之大腸桿菌BL21 StarTM (DE3) 菌落 82 圖二十七、利用IPTG誘導大腸桿菌BL21表現DENV-1 NS1重組蛋白 83 圖二十八、利用IPTG誘導大腸桿菌BL21表現DENV-2 NS1重組蛋白 84 圖二十九、利用IPTG誘導大腸桿菌BL21表現DENV-3 NS1重組蛋白 85 圖三十、 利用IPTG誘導大腸桿菌BL21表現DENV-4 NS1重組蛋白 86 圖三十一、測試培養中不同溫度下的大腸桿菌BL21中DENV-1 NS1重組蛋白表現量 87 圖三十二、在不同誘導時長的大腸桿菌BL21中DENV-1 NS1重組蛋白表現量 88 圖三十三、測試不同IPTG濃度誘導的大腸桿菌BL21中DENV-1 NS1重組蛋白表現量 89 圖三十四、經Ni-NTA樹脂純化之DENV-1 NS1重組蛋白 90 圖三十五、經Ni-NTA樹脂純化之DENV-2 NS1重組蛋白 91 圖三十六、經Ni-NTA樹脂純化之DENV-4 NS1重組蛋白 92 圖三十七、探查經DENV-2 NS1重組蛋白免疫之小鼠體內抗體效力 93 圖三十八、探查具有線性抗原決定位之抗NS1蛋白單株抗體對不同血清型登革熱病毒及茲卡病毒的特異性 95 圖三十九、利用斑點印跡法探查110個抗登革熱病毒NS1蛋白之單株抗體對日本腦炎病毒天然蛋白NS1的交叉反應 97 圖四十、利用斑點印跡法探查抗登革熱病毒NS1蛋白之單株抗體對不同血清型登革熱病毒、茲卡病毒及日本腦炎病毒天然蛋白NS1的交叉反應 98 圖四十一、利用西方墨點法探查抗登革熱病毒NS1蛋白之單株抗體與日本腦炎病毒變性蛋白NS1的交叉反應 99 圖四十二、利用西方墨點法探查抗登革熱病毒NS1蛋白之單株抗體與日本腦炎病毒變性蛋白NS1和NS1’的交叉反應 100 圖四十三、四種血清型登革熱病毒的原型毒株NS1胺基酸親緣關係樹 101 圖四十四、DENV-2 NGC strain與16681 strain NS1胺基酸之序列比對 102 圖四十五、利用斑點印跡法探查抗DENV-4變性蛋白NS1之單株抗體的特異性 103 圖四十六、日本腦炎病毒NS1、NS1’胺基酸及DENV-2 16681 strain NS1胺基酸之序列比對 104 參考文獻 105 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 登革熱 | zh_TW |
| dc.subject | 血清學鑑定 | zh_TW |
| dc.subject | 登革熱病毒 | zh_TW |
| dc.subject | NS1蛋白 | zh_TW |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | Dengue virus | en |
| dc.subject | serotyping | en |
| dc.subject | NS1 protein | en |
| dc.subject | Dengue fever | en |
| dc.subject | monoclonal antibodies | en |
| dc.title | 鑑定抗登革熱病毒非結構蛋白1的單株抗體 | zh_TW |
| dc.title | Characterization of the monoclonal antibodies against non-structural protein 1 of Dengue virus | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 顧家綺;蔡明翰 | zh_TW |
| dc.contributor.oralexamcommittee | Chia-Chi Ku;Ming-Han Tsai | en |
| dc.subject.keyword | 登革熱,登革熱病毒,血清學鑑定,NS1蛋白,單株抗體, | zh_TW |
| dc.subject.keyword | Dengue fever,Dengue virus,serotyping,NS1 protein,monoclonal antibodies, | en |
| dc.relation.page | 121 | - |
| dc.identifier.doi | 10.6342/NTU202302601 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2023-08-01 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| 顯示於系所單位： | 微生物學科所 | |
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