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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | Jing-Ru Shieh | en |
| dc.contributor.author | 謝淨如 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:38Z | - |
| dc.date.available | 2021-07-01T08:12:38Z | - |
| dc.date.issued | 2002 | |
| dc.identifier.citation | 齊肖琪。1992。傳染胰臟壞死病毒單源抗體對水產兩段核醣核酸病毒分類及缺陷性幹擾粒子之研究。國立臺灣大學動物學研究所博士論文，臺北。 郭村勇。1997。呼吸道症候群病毒致病機轉之研究。國立臺灣大學獸醫學研究所碩士論文，臺北。 林淑卿。1998。石斑魚神經壞死病毒的分離與定性。國立臺灣大學動物學研究所碩士論文，臺北。 邱顯傑。2000。石斑魚病毒神經壞死症之組織病理學與神經壞死病毒特性之研究。國立臺灣大學獸醫學研究所碩士論文，臺北。 Harlow, E.D., David Lane (1988) Antibodies, a laboratory manual. P.298 Arimoto, M., Mushiake, K., Mizuta, Y. (1992) Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish Pathology 27 (4),191-195 Arimoto, M., Mori, K., Nakai, T., Muroga, K. & Furusuwa, I. (1993) Pathogenecity of the causative agent of viral nervous necrosis disease of striped jack, Pseudocarnax dentex (Bloch& Schneider) Journal of Fish Diseases 16, 461-469 Arimoto, M., Sato, J., Maruyama, K., Mimura, G. & Furusuwa, I. (1996) Effect of chemical and physical treatment on the inactivation of striped jack nervous necrosis virus (SJNNV). Aquaculture 143, 15-22 Bloch, B., Gravningen, K., Larsen, J.L. 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Diseases of Aquatic Organisms 41, 219-223 Yoshikoshi, K., Inoue, K. (1990) Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck&Schlegel). Journal of Fish Diseases 13, 69-77 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75312 | - |
| dc.description.abstract | 以純化的石斑魚神經壞死症病毒（GNNV）作為抗原製備單源抗體，篩選出五株具有中和力價的單源抗體，分別命名為2E、9B、9D、10G以及12E；其百分之五十中和值（50％ neutralization dose, ND50）介於26-12之間；其中9B、9D、12E屬於IgM，2E、10G屬於IgG1。中和抗體9D及10G在西方墨漬法（western immunoblot）中可辨認變性（denatured）後的GNNV外鞘蛋白質，另外三株中和抗體則無反應。然而，中和抗體9D以及10G無法在酵素連結免疫吸附反應（Enzyme-linked immunosorbent assay, ELISA）中與大腸桿菌所表現的GNNV外鞘蛋白質作用，因此推測9D和10G是辨識病毒外鞘蛋白質上的醣基位置。應用中和抗體9D配合兔子抗GNNV血清，建立了double-antibody sandwich ELISA檢測系統，靈敏度為2.5-5ng／0.1ml的純化GNNV，或力價為5×103TCID50／0.1ml的細胞複製病毒上清液。根據病毒外鞘蛋白質核酸序列分析與比對，台灣所分離出的魚類神經壞死症病毒株皆屬於RGNNV基因型。利用五株中和抗體，與台灣分離病毒株進行中和反應，結果顯示，除了鯰魚病毒株與抗體2E的中和反應值較弱以外，所有台灣分離株的抗原性關係皆非常相近。中和抗體2E以及10G對四種NNV基因型病毒株皆能有效中和，而中和抗體9B、9D、12E則只能中和其中TPNNV、BFNNV及RGNNV三種基因型。利用五株中和抗體對各病毒株的中和反應結果，配合胺基酸序列之比對結果，推測抗體2E所辨識之中和抗原決定位在第124個胺基酸Valine附近。另外，推測抗體9D以及10G辨識的中和抗原決定位，在病毒外鞘蛋白質上的醣基部分。 | zh_TW |
| dc.description.abstract | Monoclonal antibodies (MAbs) against grouper nervous necrosis virus (GNNV) were established, characterized and applied for diagnosis system and analyzing the antigenic relationship of fish nodaviruses. Five MAbs (2E, 9B, 9D, 10G, 12E) with high 50% neutralizing dose (ND50) were cloned and characterized. Two of them belong to IgG1 and three of them belong to IgM. In western immunoblot assay, two MAbs (9D,10G) can react with the denatured capsid protein derived from GNNV-infected GF-1 cells, while the other three MAbs (2E, 9B, 12E) can not. MAb 9D and 10G were suggested to react with the carboxyl group of viral capsid protein due to the negative result of enzyme-linked immunosorbent assay (ELISA) which coating antigen was recombinant viral capsid protein expressed by E coli. MAb 9D accompanied with GNNV-specific rabbit antiserum were applied for developing a double-antibody sandwich ELISA system. This system can detect 2.5-5 ng / 0.1ml purified viral protein or 5×103TCID50/ 0.1ml GNNV supernatant. All Taiwan NNV isolates derived from different species of cultured fish were proved to be RGNNV genotype by comparing the nucleotide sequence of capsid protein with four NNV genotypes, and were significantly neutralized by the five MAbs except the isolate from Chinese catfish. Moreover, MAbs 2E and 10G could neutralize four NNV genotypes, but MAbs 9B, 9D and 12E could neutralize three (RGNNV, BFNNV, TPNNV). According to the results of neutralization test and the alignment of deduced amino acids of the NNV isolates, the neutralizing epitopes recognized by MAb 2E was suggested to contain the valine at location of 124th a.a.. The neutralizing epitopes recognized by MAb 9D, 10G was suggested to be carboxyl groups of capsid protein. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2002 | en |
| dc.description.tableofcontents | 誌謝…………………………………………………………………………………………………Ⅰ 目錄…………………………………………………………………………………………………Ⅱ 圖表目次……………………………………………………………………………………………Ⅳ 中文摘要……………………………………………………………………………………………Ⅴ 英文摘要……………………………………………………………………………………………Ⅵ 第一章 前言 1．病毒性神經壞死症之病史與病癥………………………………………………………………1 2．神經壞死症病毒的物、化及生物特性…………………………………………………………1 3．神經壞死症病毒的分類地位……………………………………………………………………2 4．地理分佈與宿主範圍……………………………………………………………………………3 5．流行病學（Epidemiology）之研究……………………………………………………………4 6．神經壞死症病毒散佈之病程及在宿主體內之分佈……………………………………………5 7．防疫………………………………………………………………………………………………6 8．診斷方法…………………………………………………………………………………………8 9．實驗目的…………………………………………………………………………………………9 第二章 材料與方法 1．單源抗體的製備…………………………………………………………………………………11 1．1 抗原的製備……………………………………………………………………………………11 1．2 免疫……………………………………………………………………………………………11 1．3 脾細胞收集……………………………………………………………………………………11 1．4 細胞融合………………………………………………………………………………………12 1．5 HAT篩選…………………………………………………………………………………………13 1．6 間接酵素連結免疫吸附法篩選………………………………………………………………13 1．7 單株化…………………………………………………………………………………………15 1．8 單源抗體的生產與檢定………………………………………………………………………15 1．8．1 小鼠腹水生產法……………………………………………………………………………15 1．8．2 CELLine350 flask生產法…………………………………………………………………16 1．8．3 單源抗體種類的檢定（Isotyping）……………………………………………………17 1．9 單源抗體的純化………………………………………………………………………………17 2．神經壞死症病毒重組鞘蛋白質的表現…………………………………………………………18 2．1 核酸之萃取……………………………………………………………………………………18 2．2 反轉錄－聚合?連鎖反應（RT-PCR）………………………………………………………19 2．3 重組質體的建構及蛋白質的表現……………………………………………………………20 3．單源抗體診斷系統的建立………………………………………………………………………22 3．1 病毒來源………………………………………………………………………………………22 3．2 西方墨漬法（Western immunoblot）………………………………………………………23 3．3 免疫組織化學染色（Immunohistochemistry）……………………………………………26 3．4 酵素連結免疫吸附法（Enzyme-linked immunosorbent assay, ELISA）………………28 3．5 雙抗體三明治酵素連結免疫吸附法（Double-antibody sandwich ELASA………………28 3．6 中和反應試驗（Neutralization test）……………………………………………………28 4．神經壞死症病毒分離株基因型及抗原性之分析………………………………………………29 4．1 台灣神經壞死症病毒的分離…………………………………………………………………29 4．2 核酸萃取及反轉錄－聚合?連鎖反應（RT-PCR）…………………………………………29 4．3 核酸及胺基酸序列分析………………………………………………………………………30 4．4 中和反應試驗（Neutralization test）……………………………………………………30 第三章 結果 1．單源抗體的特性分析及量產方法的比較………………………………………………………32 2．單源抗體的應用…………………………………………………………………………………33 2．1 單源抗體於免疫診斷之應用…………………………………………………………………33 2．1．1 於免疫組織化學染色之應用………………………………………………………………33 2．1．2 雙抗體三明治酵素連結免疫吸附系統之建立……………………………………………33 2．2 單源抗體於分析神經壞死症病毒分離株抗原性之應用……………………………………34 2．2．1 神經壞死症病毒台灣分離株基因型的鑑定………………………………………………34 2．2．2 神經壞死症病毒台灣分離株在抗原性的分析……………………………………………35 第四章 討論 1．單源抗體的特性…………………………………………………………………………………36 2．單源抗體應用於診斷系統的建立………………………………………………………………37 3．單源抗體應用於台灣NNV分離株的分析…………………………………………………………40 4．抗原決定位之分析………………………………………………………………………………42 5．結語與展望………………………………………………………………………………………43 第五章 參考文獻……………………………………………………………………………………44 | |
| dc.language.iso | zh-TW | |
| dc.title | 魚類神經壞死症病毒單源抗體之建立、定性及應用 | zh_TW |
| dc.title | Development, Characterization and Application of Monoclonal Antibodies against Fish Nervous Necrosis Virus | en |
| dc.date.schoolyear | 90-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 72 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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