Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3381
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dc.contributor.advisor孫幸宜zh_TW
dc.contributor.advisor王敏盈zh_TW
dc.contributor.author胡惠凌zh_TW
dc.contributor.authorHu, Hui-Lingen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T05:31:50Z-
dc.date.available2014-06-06T05:31:50Z-
dc.identifier.urihttp://hdl.handle.net/11455/3381-
dc.description.abstractIn this study, hexa-histidine tagged VP3 protein of infectious bursal disease virus (IBDV) was purified using immobilized metal ion affinity technique from the fermentation of Escherichia coli BL21 (DE3) containing a recombinant plasmid having a VP3 gene. The purification efficiencies of VP3 using Ni2+-NTA commercial agarose gels and Ni2+-IDA regenerated cellulose-based membranes at 4 C were compared, and parameters influencing the purification efficiency such as pH, imidazole concentration, and flow rate were investigated. The optimal loading condition was 20 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 6.5, whereas an efficient elution condition was 20 mM NaH2PO4, 500 mM NaCl, 500 or 750 mM imidazole, pH 7.8. By applying these conditions to the flow experiments, similar recovery (86~88%) and purity (98~99%) for VP3 were obtained in both gel column (1 ml gel) and membrane cartridge (4 membrane disks) under the flow rate of 1.7 ml/min for protein loading and 2.7 ml/min for protein elution. Regarding that the membrane process exhibited some advantages such as shorter residence time and lower cost, a better process efficiency in a large-scale system could be expected for the Ni2+-IDA membranes. Enzyme-linked immunosorbent assays (ELISAs) using the expressed VP2 or VP3 proteins of infectious bursal disease virus (IBDV) as the coating antigen for the detection of antibodies to IBDV in chickens were described in the present study. Experimental results were compared with serum neutralization and commercial-available ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rates between serum neutralization and VP2-ELISA or △TVP3-ELISA were 96.2% (251/262), and 100% (262/262), respectively, and that between serum neutralization and a commercial-available ELISA was 98.5% (257/262). At the same time, the results of the accuracy (A) and the kappa (K) value to the diagnosis system were: (VP2H-ELISA: A=95%, K=0.95; △TVP3- ELISA: A=99%, K=0.99; commercial-available ELISA: A=89%, K=0.89). The results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial-available ELISA which is generally used to replace the serum neutralization test. However, the preparation of VP3 is derived from an E. coli expression system with a high yield and purification efficiency by Ni2+-NTA gels, which is more advantage to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostics of IBDV-specific antibodies in field chickens.en_US
dc.description.abstract本研究分別利用固定化金屬親和層析(IMAC)及固定化金屬親和薄膜(IMAM)來純化傳染性華氏囊病病毒之結構蛋白VP3,並在溫度為4 C下,比較兩種不同吸附材質(Ni2+-NTA commercial agarose gels and Ni2+-IDA regenerated cellulose-based membranes)對純化效率之差異。文中探討VP3分別吸附在樹酯及薄膜上之影響因子,例如:pH值、imidazole的濃度及流速等。從實驗所得到的最適化條件為:20 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 6.5的結合緩衝溶液狀態下。而沖提條件以20 mM NaH2PO4, 500 mM NaCl, 500 or 750 mM imidazole, pH 7.8的緩衝溶液狀態下為最佳。其後,將樹酯及薄膜分別裝置於管柱和套筒中,以上述所得之最適條件做層析沖提,雖然利用這兩種不同吸附材質可以得到相似之VP3回收率(86~88%)及純度(98~99%)。但由於薄膜系統擁有較短的滯留時間及較低成本之優勢,因此在進行放大時,固定化金屬親和薄膜(Ni2+-IDA membranes)可以比固定化金屬親和層析(Ni2+-NTA gels)更廣泛的應用於工業界上。 第二階段之研究內容為探討利用在昆蟲細胞 (baculovirus)和大腸桿菌 (E.coli) 系統分別表現之重組蛋白VP2H及VP3來作為間接酵素連結免疫 吸附分析法 (indirect-ELISA) 之抗原,來發展出一套快速、靈敏和專一性 之IBD檢測方法。並將製成的VP2H- and VP3-based ELISA 套組應用於田間雞隻血清抗體之檢測。其檢測的結果與商業型檢測試劑套組 (IDEXX Kit) 和血清中和試驗結果比較,發現自製的VP2H-或△TVP3-based ELISA 套組之檢測結果與血清中和試驗相似度分別為96% (251/262) 和100% (262/262),而商業型ELISA kit與血清中和試驗相似度為98.5% (257/262)。同時,在系統準確率評估方面,本系統之準確率(Accuracy)及信賴度(kappa)各為VP2H-based ELISA (Accuracy = 95%,Kappa = 0.95) ,△TVP3-based ELISA (Accuracy = 99%,Kappa = 0.99),而IDEXX ELISA kit (Accuracy = 89%;Kappa = 0.89),因此,這表示我們的研究可以發展出ㄧ個比利用整顆病毒顆粒當作抗原之商業檢測試劑套組,更靈敏、專一且與中和抗體試驗有很好相關性及準確率之試劑套組。然而,由於大腸桿菌所表現的重組VP3蛋白,可以得到大量且亦純化的蛋白。故我們可以利用E. coli 系統所表現之重組蛋白VP3,來生產一個有效且具經濟效益的診斷IBDV抗體之ELISA套組。zh_TW
dc.description.tableofcontents頁次 中文摘要…………………………………………………………..…………...Ⅰ 英文摘要………………………………………………………………..…......Ⅲ 目錄……………………………………………………………………............Ⅴ 表目錄…………………………………………………………………..…......Ⅹ 圖目錄…………………………………………………………………..........XI 第一部分 Purification of VP3 protein of Infectious Bursal Disease Virus Using Nickel Ion-Immobilized Regenerated Cellulose-Based Membranes 1. Introduction………………………………………………………………….1 2. Experimental………………………………………………………………...3 2.1 Materials………………………………………...……………………..3 2.2 Preparation of regenerated cellulose-based IMAM…………………...3 2.3 Expression and Preparation of crude TVP3 and ΔTVP3 extract……...4 2.4 Batch purification experiments………………………………………..4 2.5 Flow purification experiments……………………………………...…5 2.6 Analysis methods…………………………………………………...…6 2.6.1 Determination of hydroxyl group content on RC membrane…...6 2.6.2 Determination of epichlorohydrin and IDA densities on RC membrane……………………………………………………….6 2.6.3 Determination of nickel ion capacity…………………………...6 2.6.4 Determination of total protein and recombinant VP3 protein amounts………………………………………………………....7 2.6.5 SDS-PAGE and Western blotting analysis……………………..7 2.6.6 Gel filtration…………………………………………………….8 2.6.7 ELISA…………………………………………………………..8 3. Results and discussion……………………………………………………..10 3.1 Regenerated cellulose-based IMAMs………………………………...10 3.2 Purification performance in the batch process………………………..12 3.2.1 Selection of buffers………………………………………...…13 3.2.2 Nonspecific binding…………………………………………..14 3.3 Purification performance in the flow process ………………………...15 3.3.1 Effect of flow rate…………………………………………….15 3.3.2 Regeneration of the immobilized metal ion affinity matrices..16 3.4 Characterization of purified VP3 protein……………………………...17 3.4.1 Native size……………………………………………………..17 3.4.2 Effect of nickel ion leakage……………………………………18 4. Conclusions……………………………………………………………...…20 5. Acknowledgement……………………………………………………….…21 6. References………………………………………………………………….22 第二部分 雞傳染性華氏囊病病毒檢測試劑組之開發 1. 前言…………………………………….…………………………………..33 2. 文獻回顧……………………………………..…………………………….35 2.1傳染性華氏囊病……………………………………..……………...….35 2.2傳染性華氏囊病病毒…………………………..……………………....35 2.3 IBDV之基因體……………………………………..……………….....36 2.4 IBDV基因體所轉譯出之VP2和VP3蛋白與其功能……………….36 2.4.1 結構蛋白-VP2…………………………………….……...…...36 2.4.2 結構蛋白、核酸結合蛋白-VP3……………...…...…….…....37 2.5 檢測IBDV之技術回顧………………………………....…..……...…38 3. 材料與方法……………………………………….……………………......42 3.1 菌種與質體………………………………………………………...…..42 3.2 蛋白質之表現及取得…………………………………………...……..42 3.3 蛋白之純化………………………………………………………...…..42 3.4 蛋白質的分析……………………………………………………...…..43 3.4.1 SDS-聚丙烯醯胺凝膠電泳………………..……………...……43 3.4.2 西方墨點法……………………………………………...……...43 3.4.3 硝酸銀染色法…………………..……………………...……….44 3.5 蛋白質定量……..………………………………………………….......45 3.5.1 總蛋白定量法………………………………………………......45 3.5.2 TVP3或△TVP3蛋白定量之方法……………………………..45 3.6 酵素連結免疫吸附實驗.………..…………………………………......46 3.6.1 IBDV抗體陽性血清來源……………………………………...46 3.6.2 VP2抗體陽性血清及VP3抗體陰性血清來源……………….46 3.6.3 IBDV抗體陰性血清來源………………………………….......46 3.6.4 田間雞隻血清來源…………………………………....………..46 3.6.5 IDEXX Kit檢測數據的取得及偵測……………………..........47 3.6.6 檢測用抗原…………………………………………………......47 3.6.7 VP2-ELISA和VP3-ELISA之製備….……………..…….…...47 3.6.8 以血清中和抗體試驗法確認陽性血清…………….…...……..48 4. 結果與討論…………………….……………………………….......……...50 4.1 重組VP3蛋白之純化與確認………………………………………....50 4.2 利用重組VP3蛋白偵測IBDV-specific抗體…………………….…..50 4.3 重組VP2H和VP3蛋白最適偵測IBDV-specific抗體之濃度…........51 4.4 VP2H-ELISA或△TVP3-ELISA與商業型的檢測試劑套組之比較….52 4.5 VP2-ELISA,VP3-ELISA或 IDEXX kit與血清中和抗體試驗檢測結 果之比較及系統辨識率的評估……………………………...…….…..52 5. 結論………………………………………….……………………...….…..55 6. 參考文獻…………...…….………………………………………………...58zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系zh_TW
dc.subject固定化金屬親和層析zh_TW
dc.subject固定化金屬親和薄膜zh_TW
dc.subject傳染性華氏囊病病毒zh_TW
dc.titlePurification of VP3 Protein of Infectious Bursal Disease Virus Using Immobilized Nickel Ion Affinity Techniques and Its Relative Applicationen_US
dc.title以固定化金屬親和薄膜純化傳染性華氏囊病病毒之結構蛋白VP3及其應用zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.grantfulltextnone-
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