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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林大盛 | |
| dc.contributor.author | Kun-Chao chang | en |
| dc.contributor.author | 張昆照 | zh_TW |
| dc.date.accessioned | 2021-06-13T02:11:07Z | - |
| dc.date.available | 2008-07-03 | |
| dc.date.copyright | 2007-07-03 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-06-23 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30646 | - |
| dc.description.abstract | 目前，豬環狀病毒二型（porcine circovirus 2; PCV2）在豬場是與離乳豬多系統性消耗症候群有關的一種重要緊急病原。此病常發生於5-12週齡豬隻。 臨床上，發生離乳豬多系統性消耗症候群（post-weaning multisystemic wasting syndrome；PMWS）症狀的豬隻，呈現體重持續下降、呼吸道症狀及偶爾可見到黃疸。
本研究的目的乃是發展一PCV2病毒之重組結構外殼蛋白（recombinant capsid protein; rORF2）， 以為檢測血清PCV2抗體之酵素連結免疫吸附分析法 (enzyme-linked immunosorbent assay；ELISA) 套組之抗原。根據豬環狀病毒二型的ORF2基因，從5端123鹼基對，開始合成專一引子。經由聚合酶鏈反應（polymerase chain reaction ；PCR）放大基因產物，選殖標的基因至pMAL-c2X基因載體，重組蛋白質表現於大腸桿菌。純化的重組蛋白質 (MBP-D41) 當作ELISA檢測套組之抗原，作為檢測豬環狀病毒二型抗體之用。 為了探討豬場內，PCV2抗體的產生與病毒循環情形之關係，我們將每一豬場，分成以下階段：1週齡、3-4週齡、8週齡、肉豬（70-80Kg）及母豬，每場總共收集50個血液樣本。 經由商業套組及自行開發的診斷套組，兩者檢測從野外豬場所採集的血清PCV2抗體。ELISA法的檢測結果，抗體陽性豬隻之比例為：90%（1週齡）、47.4 %（3-4週齡）、20 %（8週齡）、97.5 %（肉豬）、85 %（母豬）。出生後，1-8週抗體的分佈曲線呈下降趨勢。而在8週齡時，幾乎偵測不到PCV2的抗體，此時對病毒感染之敏感性增加。 檢測PCV2在不同豬隻生長階段之抗體結果，發現自行開發的ELISA與商業套組，並無明顯不同。這些結果顯示，重組蛋白MBP-D41是一潛在良好的ELISA檢測抗原。 | zh_TW |
| dc.description.abstract | Currently, porcine circovirus 2 (PCV2) is an important emerging pathogen associated with post-weaning multisystemic wasting syndrome ( PMWS) in pig farm. This syndrome of disease occurs commonly in 5-12-week-old pigs. Clinically, PMWS is characterized by progressive weight loss, respiratory signs, and occasional the jaundice in pigs.
The objective of this study was to develop a recombinant protein of the viral capsid protein (ORF2) as antigen of enzyme-linked immunosorbent assay (ELISA) kit for the detection of antibody to porcine circovirus type 2. Specific primers were synthesized according to PCV2 ORF2 DNA sequence starting from the 123 bp at 5’ end. The PCR amplicom were cloned into pMAL-c2X vector and expressed in E. Coli. The purified protein (MBP-D41) was used as ELISA antigen for detecting PCV2 antibody. To investigate the dynamics of PCV2 antibody production in relation to virus protection or circulation within the farm, we collected 50 blood samples from each farm at the following stages: 1 week, 3-4 weeks, 8 weeks, finishing pig (70-80 kg), and sow. Serological tests of PCV2 antibody were performed with commercial and “recombinant antigen (MBP-D41)” diagnostic kits in wild field. The average percentage of pigs with PCV2 antibody response in ELISA test was 90% (1 week), 47.4% (3-4 weeks), 20% (8 weeks), 97.5% (finishing pig) and 85% (sow), respectively. The distribution of the PCV2 antibody decreased from one to eight –week-old pigs. The level of antibody was almost undetectable at eight-week-old pigs. Consequently, the sensitivity to viral infection was found to be increased. There was no significant difference between our developing diagnostic kit and the commercial diagnostic kit for the detection of PCV2 antibody in various growth stages of pig. These results demonstrate that the MBP-D41 protein is a potential candidate as an ELISA antigen. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T02:11:07Z (GMT). No. of bitstreams: 1 ntu-96-R94629021-1.pdf: 1234896 bytes, checksum: 36eddfe51928a3b9dbaf3eab544cd98e (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 目次………………………………………………………………………I
圖次………………………………………………………………………V 表次……………………………………………………………………VII 中文摘要…………………………………………………………………1 英文摘要…………………………………………………………………2 第一章 序言……………………………………………………………3 第二章 文獻探討………………………………………………………6 2-1：分類………………………………………………………….……6 2-2：病毒構造及基因組成……………………………………….……6 2-2.1：Rep gene………………………………………………….……8 2-2.2：PCV2 ORF2基因及其蛋白質………………………….………11 2-2.3：PCV2病毒顆粒結構…………………………………….…….11 2-3：PCV2 臨床症狀及PMWS的定義……………………………….…12 2-3.1：臨床症狀………………………………………………………12 2-3.2：PMWS的定義……………………………………………………13 2-3.3：非PMWS條件下與PCV2有關的疾病……………………………13 2-3.3.1豬皮膚炎及腎病症候群（Porcine dermatitis and nephropathy syndrome; PDNS）………………………………….…13 2-3.3.2生殖障礙（Reproductive disorders）……………………14 2-3.3.3豬呼吸道疾病複合症（Porcine respiratory disease complex; PRDC）………………………………………………………14 2-3.3.4增生性及壞死性肺炎（Proliferative and necrotizing pneumonia；PNP）………………………………….………………..14 2-3.3.5 先天性震顫（Congenital tremor；CT）…………………15 2-4：PCV2在母豬之影響………………………………………………15 2-5：PCV2在公豬之影響………………………………………………16 2-6：PCV2的血清學……………………………………………………16 (1)具有移行抗體之能力…………………………………………17 (2)感染病毒後兩週出現抗體……………………………………17 (3)抗體力價至初生後約10週齡（72日齡）降至最低點………17 2-7：PCV2 與其他病原共同感染的影響………………………….18 2-7.1：豬肺炎黴漿菌（M.hyopneumoniae）的混合感染………….18 2-7.2：豬小病毒（porcine parvovirus）的混合感染……………18 2-7.3：其他病原的混合感染…………………………………………19 2-8：PCV2重組蛋白質表現……… ………………………….……..20 2-9：檢測方法…………………………….……………………….…20 血清交叉反應……………………………………………….21 2-10：病毒結構與免疫系統之關係………………………………….21 2-11：PCV2在公共衛生的角色…………………………………….…22 第三章 材料與方法………………………………………………….23 3-1：豬環狀病毒二型病毒（PCV2）DNA之粹取………..………….23 3-2：豬環狀病毒二型病毒（PCV2）之ORF2之載體選殖…..………23 3-3：DNA之純化………………………….……………...………….25 3-4：MBP融合蛋白質之純化……….…………………………………26 3-5：切融合蛋白質……………………………………………………27 3-6：通過直鏈澱粉膠（Amylose gel）管柱，去除大部分MBP 蛋白 ………………………………………………………………………….27 3-7：ELISA 實驗步驟…………………………………………………28 3-7.1：定量測試盤試驗 (checkboard titration)……………….28 3-7.2：ELISA操作步驟…………………………………….…………29 3-7.2.1 商業用套組ELISA方法…………………………… ……….29 3-7.2.2 自行開發之抗原（MBP-D41）ELISA方法………….………29 3-7.3：商業用套組ELISA 之模式…………………………….…….31 3-7.4：自行開發之抗原（MBP-D41）ELISA 模式………………….32 3-8：野外血清的收集…………………………………………………33 3-8.1： A 豬場……….………………………………………………33 3-8.2： B 豬場 ………………………………………………………33 第四章 實驗結果……………………………………………….……34 4-1：聚合酶鏈反應檢測及放大選殖PCV2-D41 DNA 片段….......34 4-2：經轉殖至大腸桿菌表現融合蛋白質之結果................35 4-3：經蛋白質酵素處理後，通過管柱純化後之結果............40 4-4：ELISA 檢查盤之結果………………………..………......…42 4-5：採集野外不同生長階段豬血清之PCV2抗體ELISA檢測結果...44 4-5.1：各豬場採集不同階段之血清PCV2抗體結果…………..……45 4-5.2：豬血清PCV2之ELISA抗體陽性比例……………...………..50第五章 討論…………….………………………………………....53 5-1：造成PMWS需要其他因子……………………………….……….53 5-2：豬隻在8-10週齡是危險或發病期………………………………53 5-3：病毒結構蛋白ORF2蛋白之完整結構-氨基酸序列屬於不穩定重組蛋白……………………………………………….……………....54 5-4：良好管理的豬場可以減少PCV2造成之損失率……………..…56 5-5：推測豬環狀病毒二型之可能致病機序……………………....56 第六章 參考文獻……………...…………………………………..60 附錄…...………………………………………………………………68 附錄一：重組蛋白MBP-D41之質體DNA序列……………….…………68 附錄二：重組蛋白MBP-D41之氨基酸序列……………………………73 圖1. PCV1 病毒線性基因圖…………………………………………10 圖2. PCV病毒複製起點之莖狀結構圖………………………………11 圖3. PCV2-3D電腦模擬圖……………………………………………13 圖4. 融合蛋白（MBP-D41）表現載體之架構圖……………………24 圖5. 商業用套組ELISA 之模式…………………………………….31 圖6. 自行開發之抗原（MBP-D41）ELISA 模式……………………32 圖7. 聚合酶鏈反應（PCR）放大PCV2 ORF2-D41 DNA片段圖…….34 圖8-A.經轉型至大腸桿菌BL21表現融合蛋白質之結果-SDS PAGE膠片染色.....................................................36 圖8-B.經轉型至大腸桿菌BL21表現融合蛋白質之結果(西方轉漬)- 兔抗MBP多源抗體2000倍染色…………………………………………37 圖8-C.經轉型至大腸桿菌BL21表現融合蛋白質之結果(西方轉漬)- 抗ORF2-Peptide 2號兔子抗體，2000倍染色……………………….38 圖8-D.經轉型至大腸桿菌BL21表現融合蛋白質之結果(西方轉漬)- 小鼠抗6XHis Tag 單株抗體，2000倍染色………………………….39 圖9. 經由蛋白質酵素（Factor Xa）處理後之SDS-PAGE 圖…….41 圖10. 定量最適當抗原濃度（MBP抗原檢查盤）…………………..42 圖11. 定量最適當抗原濃度（MBP-D41抗原檢查盤）………………43 圖12. 應用MBP-D41當作抗原，比較A和B豬場不同生長階段之 PCV2抗體分佈圖…………………………………………….…………48 圖13. 商業用套組當作抗原，比較A和B豬場不同生長階段之PCV2抗體分佈圖…...............................................49 圖14. A豬場之ELISA抗體陽性比例圖……………………………….50 圖15. B豬場之ELISA抗體陽性比例圖……………………………….51 圖16. 平均血清PCV2抗體陽性之百分比圖………………………….52 圖17. 豬環狀病毒二型之ORF2蛋白N端41個氨基酸二級構造圖……55 圖18. 豬環狀病毒二型之致病機序概略圖…………………………59 表1. 豬環狀病毒二型主要基因及轉錄蛋白質之基本資料….……10 表2. A場之ELISA結果(檢測抗原為MBP-D41及商品套組兩種)……45 表3. B場之ELISA結果(檢測抗原為MBP-D41及商品套組兩種)……47 表4. 每一豬場不同生長階段之平均ELISA OD值結果…………….48 表5. A及B豬場在不同生長階段之PCV2 ELISA抗體陽性比例….…50 表6. AB兩場及兩種方法之平均血清PCV2抗體陽性百分比….……52 | |
| dc.language.iso | zh-TW | |
| dc.subject | 豬環狀病毒二型 | zh_TW |
| dc.subject | 豬生殖及呼吸道症候群病毒 | zh_TW |
| dc.subject | 豬皮膚炎腎病症候群 | zh_TW |
| dc.subject | 間接免疫螢光分析法 | zh_TW |
| dc.subject | 麥芽糖結合蛋白質 | zh_TW |
| dc.subject | 離乳豬多系統性消耗症候群 | zh_TW |
| dc.subject | 酵素連結免疫吸附分析法 | zh_TW |
| dc.subject | 免疫過氧化氫單層細胞分析法 | zh_TW |
| dc.subject | indirect flurorescence immunoassay（IFIA） | en |
| dc.subject | immunoperoxidase monolayer assay（IPMA） | en |
| dc.subject | enzyme-linked immunosorbentassay（ELISA） | en |
| dc.subject | porcine reproductive and respiratory syndrome virus（PRRS） | en |
| dc.subject | porcine dermatitis and nephropathy（PDNS） | en |
| dc.subject | post-weaning multisystemic wasting syndrome（PMWS） | en |
| dc.subject | porcine circovirus type 2 | en |
| dc.subject | maltose-binding protein | en |
| dc.title | 開發豬環狀病毒二型血清抗體之酵素連結免疫吸附分析法 | zh_TW |
| dc.title | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) For Serological Diagnosis of Porcine Circovirus Type 2 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐悠深,廖明輝 | |
| dc.subject.keyword | 豬環狀病毒二型,離乳豬多系統性消耗症候群,酵素連結免疫吸附分析法,豬生殖及呼吸道症候群病毒,豬皮膚炎腎病症候群,間接免疫螢光分析法,免疫過氧化氫單層細胞分析法,麥芽糖結合蛋白質, | zh_TW |
| dc.subject.keyword | porcine circovirus type 2,post-weaning multisystemic wasting syndrome（PMWS）,enzyme-linked immunosorbentassay（ELISA）,porcine reproductive and respiratory syndrome virus（PRRS）,porcine dermatitis and nephropathy（PDNS）,indirect flurorescence immunoassay（IFIA）,immunoperoxidase monolayer assay（IPMA）,maltose-binding protein, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-06-26 | |
| dc.contributor.author-college | 生物資源暨農學院 | 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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