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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 郭應誠(Ing-Cherng Guo) | |
| dc.contributor.author | Yang-Yan Lin | en |
| dc.contributor.author | 林洋演 | zh_TW |
| dc.date.accessioned | 2021-06-08T06:00:36Z | - |
| dc.date.copyright | 2007-07-31 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-27 | |
| dc.identifier.citation | 李龍湖、蘇育平等。疫苗發展技術與實驗。動物基因轉殖與疫苗發展技術教學資源中心，2003。
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John Wiley & Sons Inc, New York. 2006 Nie Z, Tzeng YK, Chang HC, Chiu CC, Chang CY, Chang CM, Tao MH. Microscopy-based mass measurement of a single whole virus in a cylindrical ion trap. Angew Chem Int Ed Engl 45: 8131-8134, 2006 Orange N, Devauchelle G. Monoclonal antibodies against Chilo iridescent virus (iridovirus type 6). Brief report. Arch Virol 90: 349-353, 1986. Orange N, Guerillon J, Devauchelle G. Identification of viral antigenic determinants by monoclonal antibodies directed against Chilo iridescent virus (iridovirus type 6). Brief report. Arch Virol 99: 243-248, 1988. Qin QW, Lam TJ, Sin YM, Shen H, Chang SF, Ngoh GH, Chen CL. Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina. J Virol Methods 98: 17-24, 2001. Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 27: 493-497, 1938. Schnitzler P, Darai G. Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus. J Gen Virol 74: 2143-2150, 1993. Shi C, Wei Qi, Gin KYH, Lam TJ. Production and characterization of monoclonal antibodies to a grouper iridovirus. J Virol Methods 107: 147-154, 2003. Song WJ, Qin QW, Qiu J, Huang CH, Wang F, Hew CL. Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis. J Virol 78:12576-12590, 2004. Stohwasser R, Raab K, Schnitzler P, Janssen W, Darai G. Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction. J Gen Virol 74: 873-879, 1993. Tajbakhsh S, Lee PE, Watson DC, Seligy VL. Molecular cloning, characterization, and expression of the Tipula iridescent virus capsid gene. J Virol 64: 125-136, 1990. Tan WG, Barkman TJ, Gregory Chinchar V, Essani K. Comparative genomic analyses of frog virus 3, type species of the genus Ranavirus (family Iridoviridae). Virology 323: 70-84, 2004. Tapiovaara H, Olesen NJ, Lindén J, Rimaila-Pärnänen E, von Bonsdorff CH. Isolation of an iridovirus from pike-perch Stizostedion lucioperca. Dis Aquat Organ 32: 185-193, 1998. Tidona CA, Darai G. The complete DNA sequence of lymphocystis disease virus. Virology 230: 207-216, 1997. Tsai, CT, Ting JW, Wu MH, Wu MF, Guo IC, Chang CY. Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses. Virology 79: 2010-2023, 2005. Webby RJ, Kalmakoff J. Sequence comparison of the major capsid protein gene from 18 diverse iridoviruses. Arch Virol 143: 1949-1966, 1998. Williams T. Iridoviridae. Curr Top Microbiol. Immunol 116: 1-173, 1985. Zhang QY, Xiao F, Xie J, Li ZQ, Gui JF. Complete genome sequence of lymphocystis disease virus isolated from China. J Virol 78: 6982-6994, 2004. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25029 | - |
| dc.description.abstract | 石斑魚虹彩病毒（grouper iridovirus, GIV）基因體為140 Kb，有120個開放讀架（ORF）。以反向疫苗學（Reverse vaccinology）方式，利用小鼠及石斑魚抗血清，以西方轉漬來分析虹彩病毒具有抗原性之病毒蛋白，其結果發現大小約為60 kDa和45 kDa的病毒蛋白有明顯的訊號，參考虹彩病毒基因體的資訊，預測45 kDa蛋白質可能為主要鞘蛋白（major capsid protein, MCP），由基因GIV045R所轉譯，而60 kDa蛋白質則可能由GIV042L及GIV010R所轉譯。將這些目標基因利用大腸桿菌的表現系統進行其重組蛋白的表現與純化。之前實驗室已完成GIV010R (63 kDa)，GIV042L (62 kDa)的表現與純化，另外佔有病毒顆粒最大部份病毒蛋白的主要鞘蛋白GIV045R (51 kDa)，在多次全長表現遭遇困難後，予以重新設計為四個互相重疊之分段重組蛋白MCP1 (16 kDa)、MCP2 (13 kDa)、MCP3 (13 kDa)及MCP4 (11 kDa)，也順利完成其表現與純化。將此6種重組蛋白利用多種動物來源之抗GIV多株抗體，以西方轉漬法來分析此重組蛋白之抗原性，發現不同動物來源的抗體辨認到的重組蛋白之抗原性各有所不同，而對於石斑魚抗血清而言，在西方墨點法的分析下，此6種重組蛋白皆具抗原性，推測所選出的6種重組蛋白對於石斑魚可能皆具有保護的潛力，而其中又以MCP3及GIV042L，訊號較為明顯，因而此兩個重組蛋白將來會是主要的研究重點。
將這些重組蛋白，以單獨以及組合的方式，混合佐劑後腹腔內注射接種石斑魚苗進行免疫，並於21天（500 ℃天）後以力價106 TCID50 ml-1之GIV病毒液進行攻毒試驗，在38天後採血進行ELISA的分析。結果發現魚苗接種各重組蛋白後，對於GIV皆存在著一定的體液免疫力，且在接種單一重組蛋白試驗之魚苗，MCP mix及GIV042L其抗GIV力價甚至高於注射不活化GIV之正對照組，此結果與西方墨點法分析的結果部份相同，顯示所有重組蛋白皆具抗原性，且可能在接種後皆有幫助魚苗誘發體液免疫力，進而抵抗GIV感染之保護效力。 在抗體的應用方面，本研究利用兔子及山羊之抗GIV多株抗體，進行間接三明治型ELISA的開發，可以用於檢測純化後的GIV，且對於NNV則無法檢測到，而當純化之GIV稀釋到16倍（約36 ng）時還可以被檢測到，顯示此分析法具有特異性及敏感性。以目前最接近野外感染的檢體，即病毒培養液來進行偵測的結果，結果當病毒培養液（原始力價為108 TCID50 ml-1）稀釋到32倍（3.125x106 TCID50 ml-1）時還可以被檢測到，進一步評估此結果，可以應用於快速免疫分析試紙之之開發。 | zh_TW |
| dc.description.abstract | The genome of grouper iridovirus (GIV) consists 140 Kb and encodes 120 open reading frames. The results of Western blotting showed that two proteins with 60 kDa and 45 kDa, respectively, were recognized by antibodies produced with GIV viral particles. We propose the 45-kDa protein, a deduced major capsid protein (MCP), is encoded by the gene, named GIV045R, and the 60-kDa protein might be translated from the gene, named GIV042L or GIV010R. These three genes were subcloned into pET expression vector, and their recombinant proteins were produced by E. coli expression system. Because of the difficulty to effectively express MCP, we decided to divide the MCP into four segments, named MCP1, MCP2, MCP3 and MCP4 to express. The results of Western blotting showed that all the recombinant proteins have antigenicity, but compared to the others recombinant proteins, that MCP3 and GIV042L were with strong signal. These two recombinant proteins will be the target in the future experiment.
The grouper larvae were injected intraperitoneally with 2 μg of the recombinant proteins alone or combined. 21 days post-vaccination, challenged by intraperitoneally injection with 50 μl of 106 TCID50 ml-1 of GIV solution. Blood was collected and tested for ELISA after 38 days. Results of ELISA showed that all the recombinant proteins had raised titer against GIV, the group of MCP mix and GIV042L even higher than inactivated GIV-injected control group. The results showed some similarity between ELISA and Western blotting. Our results show that vaccination with the recombinant proteins may trigger the grouper larvae immune response, thus leading to protection upon live viral challeng. In this study, two polyclonal antibody against GIV from rabbit and goat were purified and used to develop an indirect sandwich ELISA for detection of GIV. The indirect sandwich ELISA could detect the purified GIV at the concentration of 36 ng and the GIV RV at the dose 3.125x106 TCID50 ml-1. Our results show that the indirect sandwich ELISA has speciality and sensitivity. These results could further be applied to immunoassay strip for the rapid diagnosis of GIV infection in grouper. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T06:00:36Z (GMT). No. of bitstreams: 1 ntu-96-R94629020-1.pdf: 825366 bytes, checksum: 5d99f89b13267a39ddd806ab6979f40c (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 目錄
中文摘要……………………………………………………….Ι 英文摘要……………………………………………………..III 目錄…………………………………………………………...V 圖次………………………………………………………...VIII 附圖……………………………………………………………..IX 第一章 緒言………………………………………………..1 第二章 文獻探討…………………………………………..3 第一節 石斑魚之簡介與台灣養殖概況……………………3 第二節 虹彩病毒…………………………………………..4 2-1 虹彩病毒之分類…………………………...4 2-2 虹彩病毒之特性…………………………...5 2-3虹彩病毒主要鞘蛋白之探討…………………6 2-4 魚類虹彩病毒感染症之相關疫情報導…….7 第三節 水產動物疫苗之簡介…………………………….9 3-1 不活化疫苗……………………………………………10 3-2 活的弱毒化疫苗………………………………….10 3-3 次單位疫苗……………………………………….11 3-4 DNA疫苗…………………………………………11 3-5 疫苗施用的方法……………………………12 3-5-1 注射劑型……………………………12 3-5-2 浸泡劑型……………………………12 3-5-3 口服劑型……………………………13 第四節 魚類虹彩病毒診斷之方法………………………13 第三章 材料與方法………………………………………15 第一節 虹彩病毒之純化…………………………………15 1-1 細胞繼代培養………………………………….15 1-2 細胞冷凍保存……………………………………15 1-3 病毒力價之測定……………………………….15 1-4 GIV之增殖……………………………………….17 1-5 GIV之純化……………………………………….17 1-6 病毒蛋白質之電泳分析……………………….18 1-7 病毒蛋白質之定量………………………………20 1-8 抗GIV抗血清之製備…………………………….21 1-8-1 免疫計畫……………………………………………..21 1-8-2 血清抗體收集………………………………………..21 1-8-3 間接型酵素免疫吸附試驗（indirect ELISA）…..…21 第二節 病毒重組蛋白的表現與應用……………………22 2-1 MCP分段重組蛋白E coli表現質體之構築…………22 2-2 勝任細胞之製備……………………………………23 2-3 轉形作用……………………………………………23 2-4 重組蛋白之誘導表現………………………………23 2-5 重組蛋白之純化……………………………………24 2-6 蛋白質定量…………………………………………24 2-7 SDS-PAGE電泳………………………………………25 2-8 西方墨點法……………………………………….25 2-9 利用魚血清進行西方墨點法…………………….26 2-10 重組蛋白接種石斑魚苗試驗………………………26 2-10-1實驗用魚…………………………………………26 2-10-2重組蛋白接種石斑魚魚苗保護效果之評估……26 2-10-3石斑魚魚苗體液免疫力ELISA分析……………….28 第三節 間接三明治型ELISA（indirect sandwich ELISA）之試驗…………...29 3-1 Protein G 親和性管柱（Protein G affinity column）之抗體純化.....29 3-2 抗體之蛋白質定量……………………………………….29 3-3 間接三明治型ELISA（indirect sandwich ELISA）……29 3-3-1 間接三明治型ELISA最佳化……………………………29 3-3-2 間接三明治型ELISA之特異性與敏感性分析……………..30 第四章 結果 …………………………………..31 第一節 虹彩病毒之增殖與純化……………………………31 1-1 GK細胞感染虹彩病毒之細胞病變效應……………31 1-2 GIV之純化……………………………………………31 1-3 GIV之SDS-PAGE電泳分析…………………………31 1-4 抗GIV之抗血清製備……………………………31 第二節 病毒重組蛋白的表現與應用………………………32 2-1 利用E. coli表現病毒重組蛋白………………...32 2-2 以不同動物抗GIV抗血清分析各重組蛋白之抗原性…....32 2-3 重組蛋白接種石斑魚苗試驗…………………….32 第三節 間接三明治型ELISA（indirect sandwich ELISA）之試驗………….33 3-1 Protein G 親和性管柱（Protein G affinity column）之抗體純化…33 3-2 間接三明治型ELISA最佳化條件………………………33 3-3 間接三明治型ELISA之特異性與敏感性分析…………34 第五章 討論………………………………………………35 參考文獻……………………………………………………..39 | |
| dc.language.iso | zh-TW | |
| dc.subject | 間接三明治型ELISA | zh_TW |
| dc.subject | 多株抗體 | zh_TW |
| dc.subject | 重組蛋白 | zh_TW |
| dc.subject | 石斑魚虹彩病毒 | zh_TW |
| dc.subject | recombinant proteins | en |
| dc.subject | indirect sandwich ELISA | en |
| dc.subject | polyclonal antibody | en |
| dc.subject | grouper iridovirus | en |
| dc.title | 石斑魚虹彩病毒之重組蛋白抗原性分析與其抗體之應用 | zh_TW |
| dc.title | Analysis of antigenic proteins of grouper iridovirus and the application of antibody | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 張繼堯(Chi-Yao Chang) | |
| dc.contributor.oralexamcommittee | 林正輝(Cheng-Hui Lin) | |
| dc.subject.keyword | 石斑魚虹彩病毒,重組蛋白,多株抗體,間接三明治型ELISA, | zh_TW |
| dc.subject.keyword | grouper iridovirus,recombinant proteins,polyclonal antibody,indirect sandwich ELISA, | en |
| dc.relation.page | 46 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2007-07-30 | |
| 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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