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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張本恆 | |
dc.contributor.author | Ching-Sen Huang | en |
dc.contributor.author | 黃靖森 | zh_TW |
dc.date.accessioned | 2021-06-16T02:36:25Z | - |
dc.date.available | 2020-07-29 | |
dc.date.copyright | 2015-07-29 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54009 | - |
dc.description.abstract | 2003 年冬天,台灣東北角養殖九孔鮑(Holiotis diversicolor supertexta)因鮑魚疱疹病毒(Abalone herpesvirus, AbHV)的感染引起大量死亡。組織病理學顯示,AbHV 主要感染神經系統,造成神經元的壞死並引發神經節神經炎。本研究的目的是表現九孔鮑魚疱疹病毒膜蛋白ORF44部分重組蛋白及製備其多株抗體,並應用於免疫組織化學染色作為診斷工具。以發病之九孔鮑組織萃取DNA,進行聚合酶鍊反應(Polymerase chain reaction, PCR)選殖膜蛋白基因並將此DNA片段經限制酶修剪後,構築外膜蛋白原核表現質體 pET 24a(+) 載體,轉形入大腸桿菌(BL21(DE3))誘導表現具有110個胺基酸之重組外膜蛋白,以IPTG誘導表現的重組蛋白質可溶於水,可以重組蛋白質C端6X-His tag之特性與Ni2+-NTA金屬離子親和性管柱進行層析純化。所得純化之重組蛋白質在SDS-PAGE蛋白質電泳分析下為13 kDa。進一步將此純化的重組蛋白質混合佐劑後,接種於兔子製備多株抗體。此多株抗體可以西方墨點轉漬法上辨認ORF44蛋白質以及在免疫組織化學染色上辨認感染鮑魚疱疹病毒之九孔神經節病變。 | zh_TW |
dc.description.abstract | Abalone herpesvirus (AbHV) infection of cultured abalone Haliotis diversicolor supertexta has induced high mortality rates in Taiwan in 2003. The histopathologic study has revealed AbHV is prone to infect neurotic tissues and has caused ganglioneuritis in moribund abalone. The purpose of this study is to express the outer membrane recombinant protein fragment ORF44 of AbHV and use this protein as an antigen to prepare the polyclone antibody, then apply in the immunohistochemistry study as diagnostic tool. We have designed a specific primers and amplified this ORF44 fragment by PCR. The amplifed DNA fragment was cleaved by restriction enzymes and inserted into pET24a (+) vector. The ORF44 expression plasmid was transformed into E. coli (BL21(DE3)) to construct the recombinant ORF44 protein with 110 amino acid residues. The IPTG-induced soluble recombinant protein was purified by a nickel-nitrilotriacetic acid (Ni2+_NTA) agarose metal ion column affinity chromatography via 6X-histidines on its C-terminal. By SDS-PAGE analysis, the molecular mass of the purified recombinant protein was about 13 kDa. The polyclonal anti-ORF44 antiserum was prepared from rabbit immunized with the adjuvant-emulsified recombinant ORF44 protein. By using the Western blotting hybridization and immunohistochemistry, the antiserum was proved to recognize the abalone hepesvirus ORF44 proteins and the neurologic lesion of AbHV-infected abalone. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:36:25Z (GMT). No. of bitstreams: 1 ntu-104-R02644006-1.pdf: 15692847 bytes, checksum: 4637658b844f62c80a23d67a6b3b452a (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………………………………………………………………………i
英文摘要…………………………………………………………………………………………………………………………ii 目錄……………………………………………………………………………………………………………………………………iii 圖次……………………………………………………………………………………………………………………………………v 第一章 前言…………………………………………………………………………………………………………………1 第二章 文獻回顧………………………………………………………………………………………………………3 第一節 軟體動物疱疹病毒之歷史背景以及流行病學………………………………3 第二節 鮑魚疱疹病毒的病理變化及致病機轉……………………………………………5 第三節 疱疹病毒科的結構…………………………………………………………………………………6 第四節 重組蛋白純化-親和性層析法之歷史背景……………………………………7 第五節 免疫組織化學染色的歷史背景…………………………………………………………8 第三章 材料與方法…………………………………………………………………………………………………10 第一節 實驗設計………………………………………………………………………………………………………10 第二節 分子生物學檢查…………………………………………………………………………………………10 3.2.1 鮑魚疱疹病毒核酸萃取………………………………………………………………………………10 3.2.2 聚合酶連鎖反應……………………………………………………………………………………………11 3.2.3 初選殖載體………………………………………………………………………………………………………12 3.2.4 次選殖載體………………………………………………………………………………………………………13 第三節 蛋白質學檢查………………………………………………………………………………………………15 3.3.1 小量培養並測試不同IPTG濃度誘導蛋白質表現………………………………15 3.3.2 大量蛋白質表現-利用Ni-NTA agarose通過管柱純化來自於大腸桿菌含6x His的重組蛋白…………………………………………………………………………………………17 3.3.3 兔子血清抗體之組織粉末之吸收……………………………………………………………18 第四節 型態病理學檢查…………………………………………………………………………………………18 3.4.1 組織病理學檢查……………………………………………………………………………………………18 3.4.2 免疫組織化學染色………………………………………………………………………………………19 第四章 結果………………………………………………………………………………………………………………20 第一節 分子生物學結果.……………………………………………………………………………………20 4.1.1 聚合酶鏈反應增幅 ORF44基因………………………………………………………………20 4.1.2 ORF44基因初選殖至TA cloning 載體pGEM®-T…………………………20 第二節 蛋白質學結果…………………………………………………………………………………………………21 4.2.1 不同表現載體(vector)之構築………………………………………………………………21 4.2.2 ORF44基因次選殖至 pET 24a表現載體…………………………………………23 4.2.3 pET24a(+)-ORF44基因蛋白質表現以及小量培養測試最佳條件…………………………………………………………………………………………………………………………………………24 4.2.4 以anti-6x-his tag抗體使用西方墨點轉漬法分析6x-his tag ORF44重組蛋白………………………………………………………………………………………………………………26 4.2.5 純化大量重組蛋白以製備兔子抗血清…………………………………………………26 4.2.6 以兔子抗血清使用西方墨點轉漬法分析ORF44重組蛋白…………………………………………………………………………………………………………………………………………27 4.2.7 以兔子抗血清使用西方墨點轉漬法分析AbHV病毒蛋白…………………27 第三節 型態病理學檢查結果…………………………………………………………………………………29 4.3.1 組織病理學檢查結果.…………………………………………………………………………………29 4.3.2 使用IHC於石蠟包埋切片……………………………………………………………………………30 第五章 討論………………………………………………………………………………………………………………34 第六章 參考文獻………………………………………………………………………………………………………40 | |
dc.language.iso | zh-TW | |
dc.title | 九孔鮑疱疹病毒ORF44部分重組蛋白之表現與應用 | zh_TW |
dc.title | Expression and application of recombinant ORF44 protein fragment of Abalone Herpesvirus | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王汎熒,陳石柱,邱品文,陳媺玫 | |
dc.subject.keyword | 鮑魚,?疹病毒,十二烷基硫酸鈉聚丙醯胺凝膠電泳,西方墨點法,免疫組織化學染色,表現, | zh_TW |
dc.subject.keyword | Abalone,Herpesvirus,SDS-PAGE,Western blotting assay,Immunohistochemistry,Expression, | en |
dc.relation.page | 51 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-27 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 分子暨比較病理生物學研究所 | zh_TW |
顯示於系所單位: | 分子暨比較病理生物學研究所 |
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