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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭益謙(Ivan-Chen Cheng) | |
| dc.contributor.author | Heng-Wei Lee | en |
| dc.contributor.author | 李恆瑋 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:20:09Z | - |
| dc.date.available | 2018-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-18 | |
| dc.identifier.citation | 甘昀騏。2012。抗口蹄疫病毒結構蛋白VP1單源抗體應用於酵素連結免疫吸附試 驗:Establishment of ELISA with monoclonal antibody against structural protein VP1 of foot-and-mouth disease virus.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49231 | - |
| dc.description.abstract | 口蹄疫可感染所有偶蹄類動物，且具有高度傳染力，在畜牧產業中是相當重要的病毒性疾病。目前，台灣的防疫政策仍然是全面施打不活化疫苗，並以血清中和試驗（serum neutralizing test, SN test）來評估免疫動物獲得中和抗體保護力的情形。然而，SN test耗時、成本高，且需在嚴格的負壓實驗室內操作。近期的研究指出，大多免疫動物的血清中以抗Site 2抗體為主。因此，我們希望以類病毒空殼蛋白（virus-like particles, VLP）和抗Site 2單源抗體（monoclonal antibody, MAb）建構阻斷型酵素連結免疫吸附試驗（blocking enzyme-linked immunosorbent assay, bELISA），並評估是否可取代SN test。VLP的製備以哺乳類細胞表現系統-transient expression assay搭配共轉染策略製造，並經過蔗醣梯度離心（sucrose gradient centrifugation）及sandwich ELISA等實驗間接確定VLP構型。為了從本實驗室已製備的41株MAbs中挑選出抗Site 2抗體，本研究採用單點突變（knock-out mutagenesis）的方式，以免疫螢光染色（immunofluorescence assay, IFA）和indirect ELISA找出可以辨識VLP但無法辨識Site 2單點突變VLP（mutated VLP, mVLP）之MAbs。配合中和抗體力價檢測結果，有6株抗Site 2抗體被篩選出來。經過sandwich ELISA以及少量樣品測試（實驗豬隻血清和臨床檢體）之bELISA，挑選S11E-9為最佳tracer完成bELISA的建構。 | zh_TW |
| dc.description.abstract | Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals worldwide. Serum neutralization test (SN test), a gold standard for evaluating the protection rate against FMDV, is still performed for disease control in Taiwan. However, SN test is laborious, expensive and requires a high-containment biosafety lab. Based on the current study on vaccinated animals, antigenic site 2 of FMDV is the most immuno-dominant neutralizing site. We aim to establish a blocking ELISA (bELISA) based on FMD virus-like particles (VLPs) and site 2 monoclonal antibody (MAb) to detect antibodies against site 2 from vaccinated animals and replace the SN test. VLPs were expressed by eukaryotic transient expression assay with co-transfect strategy and examined by sucrose gradient centrifugation accompanied with sandwich ELISA. For mapping MAb against site 2 from 41 anti-FMDV MAbs prepared previously, we performed knock-out mutagenesis with VLPs and mVLPs (site 2 mutated) by immunofluorescence assay (IFA) and indirect ELISA. Combined with neutralization assay, results indicated that 6 MAbs recognized site 2. Based on the results of sandwich ELISA and bELISA with experimental serum, S11E-9 was regarded as the best tracer to establish bELISA with VLPs. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:20:09Z (GMT). No. of bitstreams: 1 ntu-105-R03629003-1.pdf: 6858832 bytes, checksum: 9b121e9cd52764df16681c1a0ddad2f4 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 目 錄
口試委員會審定書…………………………………………………….... i 誌謝....................................................................................... ii 中文摘要……………………………………………………………….. iv 英文摘要………………………………………………….…………….. v 第一章 序言…………………………………………….…………….. 1 第二章 文獻回顧……………………………………….…………….. 4 第一節 歷史背景…………………………………......…………..…… 4 第二節 口蹄疫病毒簡介………………...…...........................………….. 5 2-2.1 IRES特性介紹............................................................. 6 2-2.2 L protease特性介紹......................................................... 7 2-2.3 3C特性介紹................................................................... 7 2-2.4 病毒結構蛋白..................................................................... 8 第三節 FMD臨床症狀、傳播方式及檢法….....…......….............. 10 2-3.1 臨床症狀............................................................................. 10 2-3.2 傳播方式............................................................................. 10 2-3.3 檢測方式........................................................................ 11 2-3.3.1 病毒分離(Virus Isolation)......................................... 11 2-3.3.2 血清中和試驗(Serum Neutralization Test)............. 11 2-3.3.3 酵素連結免疫吸附試驗(Enzyme-Link Immunosorbent__ Assay, ELISA)...................................................... 12 2-3.3.4 反轉錄聚合酶連鎖反應(Reverse Transcription-Polymerase __ Chain Reaction, RT-PCR).............................................. 12 第四節 真核細胞表現系統............................................................... 13 第五節 類病毒空殼蛋白........................................................................ 15 第六節 Epitope Mapping策略........................................................................ 18 2-6.1 Peptide-based approach......................................................... 18 2-6.2 Escape mutant assay............................................................... 19 2-6.3 Reduction of neutralizing titer........................................................... 20 2-6.4 Knock-out mutagenesis................................................................. 20 第三章 材料與方法………..……………………….……………….. 22 第一節 質體建構......................................................................... 22 3-1.1 pcDNA-VP2、pcDNA-P1、pcDNA-3C質體建構....................... 22 3-1.1.1 反轉錄作用........................................................................... 22 3-1.1.2 聚合酶鏈鎖反應（PCR）增幅VP2、P1、3C基因............. 23 3-1.1.3 切膠純化（Gel extraction）................................................... 24 3-1.1.4 Blunt-end ligation and transformation.................................. 24 3-1.1.5 Colony PCR........................................................................... 25 3-1.1.6 重組載體pJET-VP2、pJET-P1、pJET-3C之少量質體萃取. 26 3-1.1.7 重組基因置換至表現載體（pcDNA-3.1(+)）...................... 27 3-1.2 pcDNA-P1點突變實驗.............................................................................. 28 3-1.3 pcDNA-P1_2A_m3C與pcDNA-P1_2A_3C質體建構................. 30 3-1.4 pcDNA-P1_2A_mIRES_3C質體建構............................................... 31 第二節 VLP表現及抗體特性鑑定................................................................... 32 3-2.1 Transient expression assay................................................................ 32 3-2.2 免疫螢光染色（Immunofluorescene assay, IFA）......................... 33 3-2.3 VLP共轉染策略最佳化................................................................... 34 3-2.4 VLP/mVLP抗原盤製作.................................................................... 34 3-2.5 Western blot....................................................................................... 34 3-2.6 Sucrose gradient centrifugation以及VLP Ag sandwich ELISA...... 35 3-2.7 VLP/mVLP Ag Indirect ELISA（ELISA-VLP/mVLP）................... 36 3-2.8 全病毒抗原盤-O/TW/97, O/Penghu/12製作................................... 37 第三節 建立Blocking ELISA...................................................................... 37 3-3.1 單源抗體腹水之抗體純化........................................................... 37 3-3.2 純化抗體之蛋白定量................................................................ 38 3-3.3 純化抗體之HRP標示作用實驗..................................................... 38 3-3.4 最佳化VLP Ag sandwich ELISA.................................................... 39 3-3.5 初步評估抗Site 2 MAb-HRP之應用............................................. 39 3-3.6 以少量口蹄疫中和抗體陽性與陰性血清樣品評估 Site 2 MAb-HRP.................................................................... 40 3-3.7 Q10E-3與Site 2 MAb彼此間的steric effect測試..................... 41 第四節 VLP生產策略比較.................................................................... 41 3-4.1 建立Stable cell line試驗................................................................ 41 3-4.2 Transient gene expression（TGE）.................................................. 42 3-4.3 Transient expression assays與TGE搭配4種質體策略比較........ 42 第五節 其他實驗與生物材料製備.................................................................. 43 3-5.1 不活化病毒製備............................................................................. 43 3-5.2 勝任細胞製備.................................................................................. 44 3-5.3 大量質體DNA萃取...................................................................... 44 第四章 結果…………........………………………………………....... 46 第一節 以transient expression assay搭配共轉染策略製備VLP與mVLP... 46 4-1.1 RT-PCR增幅VP2、P1、3C gene.................................................. 46 4-1.2 pcDNA-VP2、pcDNA-P1、pcDNA-3C質體建構.......................... 46 4-1.3 優化3C質體轉染濃度..................................................................... 47 4-1.4 VLP/mVLP抗原盤製備.................................................................... 48 4-1.5 以Western blot確認P1被切割狀況................................................ 48 4-1.6 Sucrose gradient centrifugation-sandwich ELISA........................ 49 第二節 Site 2 MAbs及其他抗體特性鑑定................................................ 49 4-2.1 VLP/mVLP抗原盤IFA試驗（IFA-VLP/mVLP）........................... 49 4-2.2 VLP/mVLP Ag indirect ELISA試驗（ELISA-VLP/mVLP）............ 50 4-2.3 O/TW/97與O/Penghu/12全病毒抗原盤IFA試驗（IFA-O97/O12） ......................................................................................... 51 4-2.4 中和抗體力價試驗......................................................................... 51 第三節 Blocking ELISA檢測系統建立.......................................................... 52 4-3.1 Site 2 MAbs之抗體純化、定量與酵素標示（HRP conjugation）... 52 4-3.2 VLP Ag sandwich ELISA優化條件測試........................................ 52 4-3.3 初步評估抗Site 2 MAb-HRP之應用.......................................... 53 4-3.4 以少量口蹄疫中和抗體陽性與陰性血清樣品評估Site 2 MAb-HRP........................................................................................... 53 4-3.5 Site 1 MAb與抗Site 2 MAb彼此間的steric effect試驗................. 54 第四節 各項VLP生產策略評估.................................................................. 54 4-4.1 pcDNA-P1_2A_m3C及pcDNA-P1_2A_mIRES_3C質體建構..... 54 4-4.2 細胞株建立實驗結果..................................................... 55 4-4.3 Transient expression assays與transient gene expression（TGE）系 統比較.................................................................................. 55 第五章 討論…………........…………………………………….….. 57 第一節 以transient expression assay生產VLP與mVLP.............................. 58 第二節 抗體特性鑑定..................................................................... 60 第三節 Blocking ELISA建立........................................................ 63 第四節 不同表現系統及質體策略.................................................. 66 第五節 檢討..................................................................... 68 第六節 結論.................................................................. 71 參考文獻………………………………………………………….……103 附錄………………………………………………….…………….......114 圖目錄 Figure 1、口蹄疫病毒結構示意圖.............................................................................. 72 Figure 2、口蹄疫病毒之RNA序列圖........................................................................ 72 Figure 3、Cap dependent translation及Cap-independent translation示意圖.............. 73 Figure 4、FMDV IRES二級結構示意圖..................................................................... 74 Figure 5、O型口蹄疫病毒的中和抗體決定位示意圖............................................... 75 Figure 6、以PCR增幅VP2、P1、3C基因片段....................................................... 75 Figure 7、以限制酶XhoI與XbaI消化pJET-VP2、pJET-P1、pJET-3C與 pcDNA-3.1(+)........................................................................................... 76 Figure 8、VP2、P1、3C序列比對結果...................................................................... 77 Figure 9、3ABmC-C163G抗原盤測試....................................................................... 78 Figure 10、共轉染策略最佳化實驗（Transeint expression assays）......................... 79 Figure 11、共轉染實驗（Transient expression assays）之Western blot................... 80 Figure 12、Sucrose gradient centrifugation-sandwich ELISA..................................... 81 Figure 13、以mVLP-IFA鑑定篩選出疑似的抗Site 2抗體..................................... 81 Figure 14、以Indirect ELISA進行Knock-out mutagenesis實驗.............................. 84 Figure 15、O/Penghu/2012與O/TW/97之P1 amino acid sequence比對.................. 85 Figure 16、O/TW/97與O/Penghu/2012全病毒抗原盤IFA...................................... 86 Figure 17、VLP Ag sandwich ELISA條件測試........................................................ 88 Figure 18、以VLP Ag sandwich ELISA初步評估Site 2 MAb-HRP....................... 89 Figure 19、以少量血清樣品評估抗Site 2 MAb-HRP及Q10E-HRP....................... 90 Figure 20、以S11E-HRP建構之bELISA與SN titer的相關性.............................. 91 Figure 21、Q10E-3與Site 2單源抗體Steric effect測試....................................... 92 Figure 22、PCR增幅P1_2A、3C、m3C、His_3C、IRES等基因片................. 93 Figure 23、PCR增幅mIRES片段............................................................................ 93 Figure 24、以TGE製備VLP抗原盤經S11B-20 IFA結果...................................... 94 Figure 25、比較transient expression assays與TGE兩種哺乳類表現系統與四種 質體策略表現量......................................................................................... 95 Figure 26、BMAb C2 footprint.................................................................................. 96 Figure 27、Protomer示意圖........................................................................................ 97 表目錄 Table 1、41 MAbs panel特性鑑定............................................................................... 98 Table 2、共轉染策略最佳化實驗（Transeint expression assays）........................... 100 Table 3、純化抗體之蛋白濃度測定............................................................................ 100 Table 4、試驗檢體與臨床檢體之SN titer.................................................................. 101 Table 5、Site 2 MAb-HRP檢測實驗動物與臨床檢體所得R2和t值.............. 101 Table 6、Percentages of the t distribution（右單尾）................................................ 102 | |
| dc.language.iso | zh-TW | |
| dc.subject | 阻斷型酵素連結免疫吸附試驗 | zh_TW |
| dc.subject | 口蹄疫 | zh_TW |
| dc.subject | 單源抗體 | zh_TW |
| dc.subject | Site 2中和抗原決定位 | zh_TW |
| dc.subject | 類病毒空殼蛋白 | zh_TW |
| dc.subject | antigenic site 2 | en |
| dc.subject | blocking enzyme-linked immunosorbent assay | en |
| dc.subject | virus-like particles | en |
| dc.subject | monoclonal antibodies | en |
| dc.subject | foot-and-mouth disease | en |
| dc.title | 以抗Site 2單源抗體與口蹄疫類病毒空殼蛋白建構阻斷型ELISA對免疫動物進行血清學監控 | zh_TW |
| dc.title | Development of a blocking ELISA based on site 2 monoclonal antibodies and foot-and-mouth disease virus-like particles for seromonitoring vaccinated animals | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊平政,林有良,張惠雯,王金和,陳啟銘 | |
| dc.subject.keyword | 口蹄疫,單源抗體,Site 2中和抗原決定位,類病毒空殼蛋白,阻斷型酵素連結免疫吸附試驗, | zh_TW |
| dc.subject.keyword | foot-and-mouth disease,monoclonal antibodies,antigenic site 2,virus-like particles,blocking enzyme-linked immunosorbent assay, | en |
| dc.relation.page | 120 | |
| dc.identifier.doi | 10.6342/NTU201603059 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-19 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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