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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭益謙(Ivan-Chen Cheng) | |
dc.contributor.author | Yung-Hsin Lin | en |
dc.contributor.author | 林詠新 | zh_TW |
dc.date.accessioned | 2021-06-15T11:22:52Z | - |
dc.date.available | 2021-08-30 | |
dc.date.copyright | 2016-08-30 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-17 | |
dc.identifier.citation | Abdelwhab EM, Vetis J, and Mettenleiter. Avian influenza virus NS1: A small protein with diverse and versatile function. Virulence 4: 583-588, 2013.
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Zhu Q, Yang H, Chen W, Cao W, Zhong G, Jiao P, Deng G, Yu K, Yang C, Bu Z, Kawaoka Y, and Chen H. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens. J Virol 82: 220-228, 2008. Zhu X, Yu W, McBride R, Li Y, Chen LM, Donis RO, Tong S, Paulson JC, and Wilson IA. Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. Proc Natl Acad Sci USA 110: 1458-1463, 2013a. Zhu Z, Shi Z, Yan W, Wei J, Shao D, Deng X, Wang S, Li B, Tong G, and Ma Z. Nonstructural protein 1 of influenza A virus interacts with human guanylate-binding protein 1 to antagonize antiviral activity. PLoS One 8: e55920, 2013b. Zielecki F, Semmler I, Kalthoff D, Voss D, Mauel S, Gruber AD, Beer M, and Wolff T. Virulence determinants of avian H5N1 influenza A virus in mammalian and avian hosts: role of the C-terminal ESEV motif in the viral NS1 protein. J Virol 84: 10708-10718, 2010. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49303 | - |
dc.description.abstract | 禽流感病毒 (avian influenza virus, AIV)之非結構蛋白NS1,於病毒複製過程中才會產生,並不存在於病毒顆粒,因此藉由檢測血清中抗NS1抗體,可區別動物血清抗體來自於病毒感染或死毒疫苗免疫,而目前尚未有可應用的抗NS1抗體檢測工具。故本研究以H6N1 AIVs免疫BALB/c小鼠,進行融合製備抗NS1單源抗體 (αNS1 MAb),以間接免疫螢光染色法 (indirect immunofluorescence assay, IFA)、真核表現系統 (eukaryotic expression system, EES)、西方墨點法 (western blot, WB)及間接型酵素連結免疫吸附法 (indirect ELISA, iELISA)等方式進行特性鑑定。另一方面,以E. coli表現系統及桿狀病毒表現系統製備之A/chicken/Taiwan/2838V/00 (H6N1)NS1重組蛋白 (rNS1與brNS1),搭配αNS1 MAbs分別做為docking Ab與tracer Ab,篩選出最佳抗體組合 (MAb pair),最終建立以rNS1為抗原之sandwich blocking ELISA (rNS1 bELISA);此外也建立以rNS1抗原搭配αNS1 MAb做為docking Ab之sandwich ELISA (rNS1 sELISA)及直接coating rNS1之indirect ELISA (rNS1 iELISA)。以真核表現系統之IFA結果作為判定雞血清是否含抗NS1抗體之黃金標準,以三種ELISA檢測115個雞血清樣品,rNS1 bELISA的臨界值為10%,敏感性為77.8% (35/45),特異性為74.3% (52/70);rNS1 sELISA的臨界值為0.59,敏感性為93.3% (42/45),特異性為92.9% (65/70);rNS1 iELISA的臨界值為0.76,敏感性為91.1% (41/45),特異性為97.1% (68/70)。rNS1 sELISA與rNS1 iELISA具有較高之敏感性及特異性,有潛力應用於檢測雞血清中抗NS1抗體,以助於禽流感之監測與防疫。 | zh_TW |
dc.description.abstract | The non-structural protein 1 (NS1) of AIV is an important indictor Ag of AIV infection because it's not essential for formation of the viral particle but produced during viral replication. Detection of anti-NS1 antibodies in the serum can differentiate AIV infection from vaccination. Therefore, we intended to establish a blocking ELISA which enable to detect antibodies against avian influenza virus. In this study, BALB/c mice were immunized with H6N1 AIVs for generating anti-NS1 monoclonal antibodies (αNS1 MAbs). αNS1 MAbs were screened by immunofluorescence assay (IFA) and characterized by IFA, eukaryotic expression system (EES), western blot (WB), and indirect ELISA (iELISA) to identify the specificity to NS1. On the other hand, recombinant NS1 proteins (rNS1 and brNS1) from A/chicken/Taiwan/2838V/00 (H6N1) were produced by E. coli and baculovirus-insect cell expression system respectively. After optimizing the condition, we established three ELISA systems with αNS1 MAbs and rNS1, they are rNS1 blocking ELISA, rNS1 sandwich ELISA and rNS1 indirect ELISA. The IFA test of EES were taken as the golden standard method of detecting anti-NS1 antibodies in the chicken sera. 115 chicken sera were tested by three ELISA systems. The cut-off value, sensitivity and specificity of rNS1 bELISA were 10%, 77.8% (35/45) and 74.3% (52/70). The cut-off value, sensitivity and specificity of rNS1 sELISA were 0.59, 93.3% (42/45) and 92.9% (65/70). The cut-off value, sensitivity and specificity of rNS1 iELISA were 0.76, 91.1% (41/45) and 97.1% (68/70). These results show that rNS1 sELISA and rNS1 iELISA have potential to be applied on detecting serum anti-NS1 antibodies. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:22:52Z (GMT). No. of bitstreams: 1 ntu-105-R03629006-1.pdf: 14720902 bytes, checksum: c0ed137f9083acbbcc1d34cd886f1c7b (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書.........................................................................................................i
誌謝...............................................................................................................................ii 摘要.............................................................................................................................v Abstract.........................................................................................................................vi 目錄..............................................................................................................................vii 圖目錄..........................................................................................................................xii 表目錄.........................................................................................................................xiv 第一章 序言..................................................................................................................1 第二章 文獻回顧..........................................................................................................2 第一節 禽流感之歷史背景..................................................................................2 第二節 流感病毒之介紹......................................................................................2 第三節 流感病毒之複製......................................................................................4 第四節 非結構蛋白之介紹..................................................................................4 2-4.1 非結構蛋白之簡介..............................................................................4 2-4.2 N-terminal RNA binding domain (RBD) .............................................6 2-4.3 C-terminal effector domain (ED) .........................................................7 2-4.4 C-terminal tail (CTT) ...........................................................................8 2-4.5 NS1之轉譯後修飾 (Post-translational modification) ........................9 第五節 AIV之診斷方式......................................................................................9 2-5.1 AIV之抗原診斷...................................................................................9 2-5.2 AIV之抗體診斷.................................................................................10 第六節 檢測血清抗NS1抗體相關研究...........................................................10 第七節 酵素連結免疫吸附試驗........................................................................11 第三章 材料與方法....................................................................................................13 第一節 細胞培養與繼代....................................................................................13 3-1.1 MDCK細胞........................................................................................13 3-1.2 HTK細胞............................................................................................13 3-1.3 Sf9細胞...............................................................................................14 第二節 病毒培養與力價測定............................................................................14 3-2.1 病毒分離株........................................................................................14 3-2.2 病毒增殖............................................................................................14 3-2.3 血球凝集試驗 (HA test) ..................................................................15 3-2.4 病毒力價測試 (viral titration)................................. ........................15 第三節 抗禽流感病毒非結構蛋白之融合瘤單源抗體製備............................16 3-3.1 免疫計畫............................................................................................16 3-3.2 骨髓瘤細胞培養 (myeloma cell culture).........................................16 3-3.3 融合瘤製備........................................................................................16 3-3.4親代融合瘤細胞篩選.........................................................................17 3-3.5 融合瘤細胞單株化............................................................................18 第四節 重組NS1抗原之製備、純化與定量...................................................19 3-4.1 E. coli 表現系統重組NS1抗原 (rNS1)之製備、純化與定量.......19 3-4.1.1 重組NS1基因之選殖..............................................................19 3-4.1.2 重組NS1表現載體之轉型作用 (Transformation)...............19 3-4.1.3 rNS1蛋白之表現.....................................................................20 3-4.1.4 rNS1蛋白之純化與透析.........................................................20 3-4.1.5 rNS1蛋白之定量.....................................................................21 3-4.1.6 rNS1蛋白之確認.....................................................................21 3-4.2 Baculovirus表現系統重組NS1抗原 (brNS1)之製備、純化與定量 ............................................................................................................22 3-4.2.1 重組NS1基因之選殖............................................................22 3-4.2.1.1 聚合酶連鎖反應 (PCR)增幅2838/H6N1 full length NS1 gene.....................................................................................22 3-4.2.1.2 建構重組轉移載體 (Transfer vector).........................23 3-4.2.1.3 重組轉移載體PAK9-NS1之轉型作用 (Transformation)..........................................................................23 3-4.2.1.4 重組轉移載體PAK9-NS1之確認...............................24 3-4.2.1.5重組轉移載體PAK9-NS1之定序 (Sequencing)........24 3-4.2.1.6 重組轉移載體PAK9-NS1之萃取..............................25 3-4.2.2 建構重組桿狀病毒 Bac-NS1................................................25 3-4.2.2.1 共轉移感染 (Co-transfection)....................................25 3-4.2.2.2 檢出基因轉殖成功之重組病毒並增殖 (Amplification)............................................................................26 3-4.2.2.3 測定桿狀病毒力價 (50% Tissue Culture Infective Dose, TCID50).............................................................................27 3-4.2.3 重組蛋白brNS1之製備、純化與定量................................27 3-4.2.3.1 brNS1蛋白之表現.......................................................27 3-4.2.3.2 brNS1蛋白之純化與透析...........................................28 3-4.2.3.3 brNS1蛋白之定量.......................................................28 3-4.2.3.4 brNS1 蛋白之確認......................................................28 第五節 αNS1單源抗體之特性鑑定與製備......................................................28 3-5.1 免疫螢光染色法 (IFA).....................................................................28 3-5.2真核表現系統 (Eukaryotic expression system, EES).......................28 3-5.3 西方墨點法 (Western blot)...............................................................29 3-5.4 單源抗體之亞型分析 (Isotyping) ...................................................29 3-5.5 腹水製備............................................................................................29 3-5.6 單源抗體之純化與定量....................................................................30 3-5.7 單源抗體之酵素標示 (HRP-conjugation)............ ..........................30 第六節 建立αNS1 MAbs-based blocking ELISA.............................................31 3-6.1 以indirect ELISA測試αNS1 MAbs之特異性及敏感性................31 3-6.2 最佳化αNS1 MAbs-based blocking ELISA.....................................31 3-6.2.1 最佳化tracer Ab與rNS1或brNS1之比例.........................31 3-6.2.2 最佳化docking Ab、rNS1或brNS1與tracer Ab之比例...32 3-6.3 血清檢體檢測....................................................................................32 3-6.3.1 血清檢體之免疫螢光染色法 (IFA ) ....................................32 3-6.3.2 以rNS1 blocking ELISA檢測血清抗體...............................32 3-6.3.3 以rNS1 sandwich ELISA檢測血清抗體..............................33 3-6.3.4 以rNS1 indirect ELISA檢測血清抗體.................................33 3-6.3.5 rNS1 bELISA、rNS1 sELISA及rNS1 iELISA之cut-off value、敏感性及特異性分析..............................................................34 第四章 結果................................................................................................................35 第一節 抗禽流感病毒非結構蛋白之融合瘤單源抗體製備............................35 4.1.1 H6N1免疫小鼠之血清力價...............................................................35 4-1.2 親代融合瘤細胞之篩選....................................................................35 第二節 NS1抗原之製備與確認........................................................................36 4-2.1 rNS1蛋白之表現、純化與定量........................................................36 4-2.2 rNS1蛋白之確認................................................................................36 4-2.3 PCR增幅2838/H6N1 full length NS1 gene.......................................36 4-2.4 建構重組轉移載體............................................................................37 4-2.5 重組載體PAK9-NS1之確認............................................................37 4-2.6 重組載體PAK9-NS1之定序............................................................37 4-2.7 桿狀病毒力價之測定........................................................................37 4-2.8 brNS1蛋白之表現、純化、定量與確認...........................................38 第三節 αNS1單源抗體之特性鑑定與製備......................................................38 4-3.1 免疫螢光染色....................................................................................38 4-3.2 西方墨點法........................................................................................39 4-3.3 單源抗體之亞型分析........................................................................39 4-3.4 單源抗體之純化、定量與酵素標示................................................39 第四節 建立αNS1 MAbs-based blocking ELISA.............................................40 4-4.1以indirect ELISA測試αNS1 MAbs之特異性及敏感性................40 4-4.2最佳化αNS1 MAbs-based blocking ELISA......................................40 4-4.3 篩選最佳的抗體組合 (MAb pairs) .................................................41 4-4.3.1 挑選OD值較高的MAb pair.................................................41 4-4.3.2 以少量血清最佳化αNS1 MAbs-based blocking ELISA......41 4-4.3.3 以少量血清篩選最佳抗體組合.............................................42 4-4.4 最佳化sandwich ELISA及indirect ELISA.....................................42 4-4.5 血清檢體之檢測................................................................................42 4-4.5.1 血清檢體種類及免疫螢光染色 (IFA) .................................43 4-4.5.2 rNS1 bELISA、rNS1 sELISA及rNS1 iELISA之cut-off value、敏感性及特異性分析..............................................................43 第五章 討論................................................................................................................44 第一節 抗體製備與特性鑑定............................................................................44 第二節αNS1 MAbs-based ELISA之建立與測試.............................................48 參考文獻......................................................................................................................87 附錄.........................................................................................................................104 圖目錄 Figure 1、Schematic representation of NS1 protein....................................................58 Figure 2、H6N1免疫小鼠之血清力價測定..............................................................59 Figure 3、SDS-PAGE 與Western blot確認rNS1蛋白............................................60 Figure 4、PCR增幅2838/H6N1 full length NS1 gene...............................................60 Figure 5、限制酶EcoRI與XbaI切割NS1-6x His與pBacPAK9...........................61 Figure 6、Colony PCR確認NS1-6x His是否插入pBacPAK9................................61 Figure 7、NS1-6x His gene之序列比對....................................................................62 Figure 8、NS1-6x His之胺基酸序列比對.................................................................62 Figure 9、以SDA-PAGE與Western blot分析brNS1蛋白純化之效率.................63 Figure 10、αNS1 MAbs對2904/H6N1之IFA結果.................................................64 Figure 11、真核表現系統之IFA結果.......................................................................65 Figure 12、αNS1 MAbs對rNS1及brNS1之Western blot結果.............................66 Figure 13、αNS1 MAbs之亞型鑑定..........................................................................67 Figure 14、以indirect ELISA測試αNS1 MAbs之敏感性及特異性......................68 Figure 15、最佳化tracer Ab與抗原比例..................................................................69 Figure 16、比較11種αNS1 MAbs作為tracer Ab之表現......................................70 Figure 17、最佳化docking Ab、抗原與tracer Ab之比例......................................71 Figure 18、篩選最佳抗體組合 (MAb pairs) ............................................................72 Figure 19、以少量雞血清最佳化αNS1 MAbs-based blocking ELISA......................73 Figure 20、以少量雞血清篩選最佳抗體組合............................................................74 Figure 21、以少量雞血清優化sandwich ELISA及indirect ELISA之rNS1使用..75 Figure 22、以少量雞血清優化sandwich ELISA之docking Ab使用量...................76 Figure 23、三種ELISA之ROC曲線.......................................................................77 Figure 24、1209/H5N2與2838/H6N1之NS1序列比對結果.................................78 Figure 25、以SDS-PAGE分析大量rNS1與brNS1蛋白.......................................78 Figure 26、MP-5 NS1 Ab bELISA測試50個HI陽性雞血清抗NS1抗體...........79 表目錄 Table 1、Influenza viruses used in the study................................................................80 Table 2、Immunization plan of BALB/c mice.............................................................80 Table 3、Immunization plan of BALB/c mice after 1st fusion.....................................81 Table 4、Summarizing the characterization of 22 αNS1 MAbs..................................82 Table 5、Purification and quantification of αNS1 MAbs............................................83 Table 6、HRP conjugation of αNS1 MAbs..................................................................83 Table 7、以經驗法則之cut-off value計算敏感性與特異性....................................83 Table 8、以ROC曲線之cut-off value計算敏感性與特異性..................................83 Table 9、5場AIV周邊監測場採樣之99個HI陽性雞血清樣品..........................84 Table 10、5場AIV周邊監測場HI力價之幾何平均數與抗NS1抗體陽性率.....85 Table 11、The MAb pairs on NS1 Ag sandwich ELISA in previous study.................85 Table 12、Host-cell proteins that interact with the influenza A virus NS1 protein.....86 | |
dc.language.iso | zh-TW | |
dc.title | 以抗NS1單源抗體建立ELISA應用於檢測抗禽流感病毒之抗體 | zh_TW |
dc.title | Detection of antibodies against avian influenza virus by anti-NS1 monoclonal antibodies-based ELISA | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王金和,鄭明珠,趙裕展,張伯俊 | |
dc.subject.keyword | 禽流感病毒,非結構蛋白,單源抗體,酵素連結免疫吸附法, | zh_TW |
dc.subject.keyword | avian influenza virus (AIV),NS1 protein,monoclonal antibody (MAb),enzyme-linked immunosorbent assay (ELISA), | en |
dc.relation.page | 111 | |
dc.identifier.doi | 10.6342/NTU201603029 | |
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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