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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭益謙(Ivan-Chen Cheng) | |
| dc.contributor.author | Jia-Rong Wu | en |
| dc.contributor.author | 吳佳蓉 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:30:37Z | - |
| dc.date.copyright | 2019-08-18 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21301 | - |
| dc.description.abstract | 禽流感病毒(Avian influenza virus, AIV)是具有封套之負向單股RNA病毒，並有八段RNA基因片段，病毒封套上包含三種跨膜蛋白: 血液凝集素(Hemagglutinin, HA)、神經胺酸酶(Neuraminidase, NA)與基質蛋白(Matrix, M1)。AIV可以根據病毒表面蛋白HA與NA進行分型，HA可以與宿主細胞上唾液酸接收器(sialic acid receptor)連接造成感染，而NA主要功能為切割病毒與唾液酸之連結，幫助病毒出芽離開宿主細胞。台灣所發生的禽流感案例，在家禽曾發現的低病原性禽流感病毒亞型，有H6N1與H5N2，分別於1972年與2003年首次於養禽場被檢測出，可能造成人畜共通傳染病等公共衛生議題，因此發展具有特異性與敏感性之檢測工具是迫切需要的。本研究使用不活化A/chicken/Taiwan/2838V/00(H6N1) 以及A/chicken/Miaoli/2904/00(H6N1)禽流感病毒對 BALB/c小鼠進行免疫，以融合瘤技術製備辨識NA之單源抗體 (αNA MAb)，並使用免疫螢光染色法(IFA)、西方墨點法(western blotting)以及間接型酵素連結免疫吸附法(indirect ELISA)評估MAb對NA抗原之特異性與敏感性。同時製備重組NA蛋白做為抗原，並使用桿狀病毒-昆蟲細胞表現系統大量製造具有四聚體結構的NA；此外，以蔗糖梯度純化2904/H6N1全病毒，以完整病毒作為抗原。利用所製備之單源抗體與抗原(rN1和2904/H6N1)建立三明治型酵素連結免疫吸附法(sandwich ELISA)，並優化阻斷型酵素連結免疫吸附法(blocking ELISA, bELISA)之條件，建構MAb-based bELISA，並以NI test檢測受測雞血清輔助確認，期應用於能區別N1與N2亞型動物血清之檢測，協助禽流感診斷、分型以及防疫監測。初步測試顯示，以2904/H6N1為抗原，使用MAb組合61YA1-2a & 61YA1-2a-HRP，前驅測試8個分別H6陽性與陰性雞血清樣品，並搭配NI test輔助確認血清中抗體NA亞型別，在bELISA結果中皆有血清OD讀值顯著下降，NI test中有5個血清NA抗體抑制率達到50%，代表血清含有抗體抑制作用，後續有待大量血清樣品測試以顯示所建立MAb-based bELISA之效能。 | zh_TW |
| dc.description.abstract | Avian Influenza virus (AIV) is an enveloped RNA virus with eight RNA- segments. The viral envelope contains three transmembrane proteins: hemagglutinin(HA), neuraminidase(NA), and matrix. AIV subtypes can be classified by HA and NA. The major function of NA is essential to cleave the sialic receptors and viral membranes, and required for virus budding and releasing. Low pathogenic H6N1 AIV has circulated in poultry in Taiwan for 40 years and H5N2 has been isolated in Taiwan since 2003 respectively. These endemic zoonotic pathogens should potentially intimidate the public health. Therefore, to develop the specific and sensitive detection tools is a crucial need. In this study, BALB/c mice were immunized with inactivated A/chicken/Taiwan/2838V/00(H6N1) and A/chicken/Miaoli/2904/00 (H6N1) AIV for generating anti-NA monoclonal antibodies (αNA MAb). These MAbs were screened by IFA, analyzed by western blotting(WB), and indirect ELISA(iELISA) to evaluate the specificity and sensitivity to NA. We also cloned two recombinant NA from H6N1 AIV: full-length N1(rN1) and GCN4N1, and used baculovirus expression system to produce the recombinant and tetrameric NA. Meanwhile, we purified 2904/H6N1 virus by sucrose gradient and used complete virus as the antigen for ELISA. Monoclonal antibody against NA and NA antigens (rN1 and 2904/H6N1) were applied to establish two sandwich ELISAs (sELISA). The MAb-based bELISA was obtained after the condition of sELISA was optimized. Based on the result of analysis and optimization, we choose 2904/H6N1 for the antigen, and 61YA1-2a & 61YA1-2a-HRP as the MAb pair for the MAb-based bELISA. Then eight chicken sera H6 HI positive and negative chosen for primary test respectivly. All H6 positive sera showed low OD value in the bELISA test and five of them have NI activities, which means that these sera may have the blocking antibody revealed by this MAb-based bELISA. Lastly, the aim to establish a bELISA platform with 2904/H6N1 and αNA MAb/H6N1 and the efficacy the bELISA will be compared with the results of the NI test. | en |
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| dc.description.tableofcontents | 口試委員會審定書-------------------------------------------------------------------------- i
致謝-------------------------------------------------------------------------------------------- ii 中文摘要-------------------------------------------------------------------------------------- iv 英文摘要-------------------------------------------------------------------------------------- v 目錄-------------------------------------------------------------------------------------------- vi 圖目錄----------------------------------------------------------------------------------------- xii 表目錄 ---------------------------------------------------------------------------------------- xiv 第一章 序言 -------------------------------------------------------------------------------- 1 第二章 文獻回顧 -------------------------------------------------------------------------- 3 第一節 禽流感背景與起源----------------------------------------------------------- 3 第二節 流感病毒介紹----------------------------------------------------------------- 3 第三節 流感病毒感染與複製-------------------------------------------------------- 4 第四節 台灣禽流感之疫情----------------------------------------------------------- 5 第五節 神經胺酸酶(NA)介紹-------------------------------------------------------- 5 2-5.1 NA亞型分類------------------------------------------------------------------- 6 2-5.2 NA結構與功能 --------------------------------------------------------------- 6 2-5.2.1 NA結構 ----------------------------------------------------------------------- 6 2-5.2.2 NA功能 ----------------------------------------------------------------------- 7 2-5.3 NA醣基化構造與功能 ------------------------------------------------------ 7 第六節 重組蛋白NA與Leucine zipper domain GCN4 sequence -------------- 8 第七節 桿狀病毒-昆蟲細胞表現系統(Baculovirus expression system)-------------- 8 第八節 AIV診斷方式 ---------------------------------------------------------------- 9 2-8.1 AIV之抗原診斷--------------------------------------------------------------- 9 2-8.2 AIV之抗體診斷-------------------------------------------------------------- 10 第九節 酵素連結免疫吸附法(Enzyme-linked immunosorbent assay, ELISA) ---- 10 2-9.1 ELISA類型與應用------------------------------------------------------------ 10 2-9.2 競爭型或阻斷型酵素連結免疫吸附法(cELISA/bELISA)------------ 11 第三章 材料與方法-------------------------------------------------------------------------- 12 第一節 細胞培養與繼代-------------------------------------------------------------- 12 3-1.1 MDCK細胞 ------------------------------------------------------------------ 12 3-1.2 Sf9細胞 ----------------------------------------------------------------------- 12 3-1.3 HTK細胞 --------------------------------------------------------------------- 12 3-1.4 病毒分離株 ------------------------------------------------------------------ 13 3-1.5 病毒濃縮與純化 ------------------------------------------------------------ 13 第二節 免疫計畫----------------------------------------------------------------------- 13 3-2.1 免疫計畫 --------------------------------------------------------------------- 13 3-2.2 骨髓瘤細胞培養-------------------------------------------------------------- 14 3-2.3 製備融合瘤-------------------------------------------------------------------- 14 3-2.4 親代融合瘤細胞篩選-------------------------------------------------------- 15 3-2.5融合瘤細胞單株化 ---------------------------------------------------------- 15 第三節 αNA 單源抗體之特性鑑定與製備 -------------------------------------- 16 3-3.1 免疫螢光染色法(Immunofluorenscence Assay, IFA) ------------------ 16 3-3.2 真核表現系統(Eukaryotic expression system, EES) -------------------- 16 3-3.3 單源抗體亞型分析(Isotyping) --------------------------------------------- 16 3-3.4 腹水製備----------------------------------------------------------------------- 17 3-3.5 腹水純化及定量-------------------------------------------------------------- 17 3-3.6 單源抗體酵素標示(HRP-conjugation) ----------------------------------- 18 第四節 製備重組rNA/H6N1抗原-------------------------------------------------- 18 3-4.1 重組rNA/H6N1基因之選殖----------------------------------------------- 18 3-4.1.1 RNA萃取---------------------------------------------------------------------- 18 3-4.1.2 反轉錄作用-------------------------------------------------------------------- 18 3-4.1.3 聚合酶連鎖反應(PCR)增幅2838/H6N1 full-length NA gene----------------- 19 3-4.1.4 Blunt-end cloning -------------------------------------------------------------- 20 3-4.1.5 重組載體pJET1.2-N1轉型(Transformation) --------------------------------- 21 3-4.1.6 Colony PCR -------------------------------------------------------------------- 21 3-4.1.7 重組載體pJET1.2-N1定序(Sequencing) ------------------------------------- 22 3-4.2 建構重組轉移載體----------------------------------------------------------- 22 3-4.2.1聚合酶連鎖反應(PCR)增幅N1/H6N1、∆N1/H6N1 gene--------------------- 22 3-4.2.2 建構重組轉移載體(Transfer vector) ------------------------------------------ 25 3-4.2.3 重組載體pcDNA-N1、pBac-N1、pcDNA-∆N1、pBac-∆N1轉型之作用(Transformation)--26 3-4.2.4 Colony PCR-------------------------------------------------------------------- 26 3-4.2.5 重組轉移載體pcDNA-N1 & pBac-N1之定序(Sequencing) ----------------- 26 3-4.2.6 重組載體pBac-GCN4N1與pcDNA-GCN4N1建構-------------------------- 26 3-4.2.7 重組載體pBac-GCN4N1與pcDNA-GCN4N1轉型之作用(Transformation) -------27 3-4.2.8 Colony PCR -------------------------------------------------------------------- 27 3-4.2.9 重組轉移載體pBac-GCN4N1與pcDNA-GCN4N1之定序(Sequencing) ---- 27 3-2.2.10 重組載體pcDNA-N1、pcDNA-GCN4N1、pBac-N1、pBac-GCN4N1之大量萃取---- 28 3-4.3 重組桿狀病毒的建構-------------------------------------------------------- 28 3-4.3.1共轉移感染(Co-transfection) -------------------------------------------------- 28 3-4.3.2轉殖成功重組病毒之增殖(Amplification) ------------------------------------ 29 3-4.3.3 測定桿狀病毒力價(50% Tissue Culture Infective Dose, TCID50) ------------- 30 3-4.4 重組蛋白Bac-N1 or Bac-GCN4N1製備、純化與定量---------------- 30 3-4.4.1 Bac-N1 or Bac-GCN4N1蛋白表現--------------------------------------------- 30 3-4.4.2 Bac-N1 or Bac-GCN4N1蛋白濃縮--------------------------------------------- 31 3-4.4.3 Bac-N1蛋白純化--------------------------------------------------------------- 31 3-4.4.4 Bac-GCN4N1蛋白純化-------------------------------------------------------- 31 3-4.4.5 Bac-N1與Bac-GCN4N1蛋白質定量------------------------------------------ 32 3-4.4.6 Bac-N1與Bac-GCN4N1蛋白確認--------------------------------------------- 32 第五節 建立αNA MAbs-based blocking ELISA ----------------------------------- 33 3-5.1 以indirect ELISA測試αNA MAbs之特異性與敏感性-------------- 33 3-5.2 最佳化αN1NA MAbs-based blocking ELSIA---------------------------- 33 3-5.2.1最佳化tracer Ab 與N1抗原之比例------------------------------------------- 33 3-5.2.2 最佳化docking MAb、N1抗原與tracer Ab之比例-------------------------- 34 3-5.3血清檢體檢測------------------------------------------------------------------ 34 3-5.3.1 NA酵素活性抑制試驗(NA inhibition test, NI test) ---------------------------- 34 3-5.3.2抗原N1與H6N1 blocking ELISA 檢測血清檢體---------------------------- 34 3-5.3.3 rN1/H6N1與2904/H6N1 bELISA之cut-off value與敏感性、特異性分析--- 35 第四章 結果----------------------------------------------------------------------------------- 36 第一節 rN1/H6N1、GCN4N1/H6N1抗原製備與確認-------------------------- 36 4-1.1 重組rN1/H6N1、GCN4N1/H6N1 基因選殖----------------------------- 36 4-1.1.1 以PCR增幅出2838/H6N1 full-length NA gene------------------------------- 36 4-1.1.2 重組載體pJET1.2-N1之確認------------------------------------------------- 36 4-1.1.3 重組載體pJET1.2-N1之定序------------------------------------------------- 36 4-1.1.4 PCR增幅2838/H6N1 full-length NA gene與truncated NA gene-------------- 36 4-1.1.5 建構重組轉移載體------------------------------------------------------------ 36 4-1.1.6 重組載體pcDNA-N1、pcDNA-GCN4N1、pBac-N1、pBac-GCN4N1定序確認-----37 4-1.2 重組蛋白桿狀病毒力價之測定(TCID50) -------------------------------- 37 4-1.3 Bac-N1與Bac-GCN4N1抗原盤之免疫螢光染色---------------------- 37 4-1.4 rN1/H6N1與rGCN4N1蛋白質濃縮、純化與定量--------------------- 38 第二節 抗禽流感病毒N1NA之融合瘤單源抗體製備-------------------------- 38 4-2.1 2838/H6N1、2904/H6N1免疫小鼠之血清力價-------------------------- 38 4-1.2 N1親代融合瘤細胞篩選---------------------------------------------------- 39 第三節 αN1NA單源抗體之特性鑑定與製備------------------------------------- 39 4-3.1 免疫螢光染色(IFA) --------------------------------------------------------- 39 4-3.2 rN1與rGCN4N1西方墨點法(western blotting)以及抗原構型確認- 40 4-3.3 單源抗體之亞型分析(Isotyping) ------------------------------------------ 40 4-3.4 單源抗體純化、定量以及酵素標示(HRP-conjugation) --------------- 40 第四節 建立αNA MAbs-based blocking ELISA---------------------------------- 41 4-4.1 以indirect ELISA測試αNA MAbs之特異性與敏感性--------------- 41 4-4.2 最佳化αNA MAbs based blocking ELISA條件------------------------- 41 4-4.3 挑選最佳抗體組合(MAbs pairs) ------------------------------------------ 42 4-4.3.1 挑選OD值較高的MAbs pairs------------------------------------------------ 42 4-4.3.2 以少量雞血清最佳化αN1 MAbs-based blocking ELISA---------------------- 42 4-4.3.3 以bELISA與IFA測試雞血清之敏感性與特異性分析---------------------- 43 4-4.4搭配NI test測試待測雞血清中所含NA抗體-------------------------- 43 第五章 討論----------------------------------------------------------------------------------- 44 第一節rN1與GCN4N1蛋白表現--------------------------------------------------- 44 第二節 抗體特性鑑定分析----------------------------------------------------------- 45 αN1 MAbs特性鑑定--------------------------------------------------------- 46 第三節 αNA MAbs-based blocking ELISA建立與測試------------------------- 48 5-3.1 篩選最佳抗體組合----------------------------------------------------------- 48 第六章 結論----------------------------------------------------------------------------------- 51 參考文獻--------------------------------------------------------------------------------------- 82 附錄--------------------------------------------------------------------------------------------- 87 | |
| dc.language.iso | zh-TW | |
| dc.title | 應用流感病毒四聚體神經胺酸酶藉由阻斷型ELISA檢測抗H6N1抗體 | zh_TW |
| dc.title | The application of tetrameric influenza virus neuraminidase for detection of antibodies against H6N1 by blocking ELISA | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王金和,鄭明珠,陳慧文,鄭達駿 | |
| dc.subject.keyword | 禽流感病毒,神經胺酸?,單源抗體,阻斷型酵素連結免疫吸附法, | zh_TW |
| dc.subject.keyword | Avian influenza virus,neuraminidase,monoclonal antibody,blocking ELISA, | en |
| dc.relation.page | 92 | |
| dc.identifier.doi | 10.6342/NTU201903477 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-08-14 | |
| 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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