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研究生:鄭筑帆
研究生(外文):Chu-Fan Cheng
論文名稱:石斑魚虹彩病毒ORF5L及ORF120L基因的特性分析與抗體製備
論文名稱(外文):Characterization and antibody production of two late gene encoded by grouper iridovirus ORF5L and 120L
指導教授:賴裕順
指導教授(外文):Yu-Shen Lai
口試委員:邱品文呂明偉
口試委員(外文):Pin-Wen ChiouMing-Wei Lu
口試日期:2017-01-20
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術與動物科學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:65
中文關鍵詞:石斑魚紅彩病毒GIV-5LGIV-120L晚期基因抗體製備
外文關鍵詞:grouper iridovirusGIV-5LGIV-120Llate geneantibody production
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石斑魚虹彩病毒(grouper iridovirus, GIV)分類上屬於虹彩病毒科(Iridoviridae)、蛙虹彩病毒屬(Ranavirus)的大型DNA病毒,基因體為139,793 base pair (bp),預測有120 ORFs (open reading frames)。目前已知GIV是全世界重大經濟養殖魚的重要致病菌之一,因此為了早期診斷、治療和預防病毒感染,病毒基因的功能角色必需被清楚知道。本研究目的主要探討GIV-5L及120L基因在病毒感染過程的特性分析。

首先將GIV-5L及120L於NCBI資料庫進行序列分析,初步推測皆為Ranavirus特有的病毒蛋白,而GIV-120L則可能為neurofilament triplet H1-like protein。以GIV基因體為模板配合專一性引子進行PCR反應,可以得到GIV-5L基因969 bp及120L基因1,470 bp,接著分別將這兩個基因各別構築到pET-23a表現載體,再轉形至Escherichia coli BL21 (DE3),IPTG誘導後,大量重組蛋白經鎳親和性管柱純化後,分別得到分子量37.8 kDa (kilo dalton)的 GIV-5L-His重組蛋白及分子量59.1 kDa的GIV-120L-His重組蛋白,接著利用這些重組蛋白當免疫原進行小鼠免疫,八週後取小鼠脾臟與骨髓瘤細胞(SP2/O myeloma cells)進行融合試驗,分別得到GIV-5L的多株抗體及GIV-120L的單株抗體。

GIV-5L及120L基因表現特性分析,在GIV 以multiplicity of infection (MOI) = 5感染GK (grouper kidney)細胞時,利用cycloheximide (CHX)和cytosine arabinoside (AraC)來進行GIV-5L及120L基因表現時期分析,確認GIV-5L轉錄體表現時間為9到30 h post-infection (p.i.),GIV-120L則為12到30 h p.i.,GIV-5L蛋白體表現時間為9到30 h p.i.,GIV-120L則為感染後18到30 h p.i.,並且兩者皆被CHX及AraC抑制,確認為晚期基因。接著利用已製備好的抗體進行免疫螢光染色分析,確認GIV-5L蛋白表現位置在12 h p.i.位於細胞質,24 h p.i.則位於細胞核,GIV-120L在24 h p.i.位於病毒組裝位置(virus assembly site)。本研究結果提供GIV-5L及120L病毒基因的功能特性,有助於了解GIV的組裝方式以及GIV診斷之應用。

Grouper iridovirus (GIV), a large DNA virus, belongs to the genus Ranavirus of the family Iridoviridae. The genome was 139,793 base pair (bp) in length and contained 120 predicted open reading frames (ORFs). GIV is one of the most important viral pathogens in aquaculture fish and cause economic losses. To understand the molecular mechanism of the pathogenesis, this study identified GIV-5L and 120L by using bioinformatics and proteomic methods

Alignment of amino acid sequences was carried out using NCBI BLAST. The homologs of GIV-5L and 120L are only in the genus Ranavirus. GIV-120L contains a conserved domain of neurofilament triplet H1-like protein. GIV-5L and 120L are 969 and 1,470 bp in length. The full-length gene was amplified by PCR and cloned into expression vector. Escherichia coli BL21 (DE3) cells was transformed with the recombinant plasmids and induced by IPTG. The recombinant protein, 37.8 kDa (kilo Dalton) and 59.1 kDa, of GIV-5L-His and 120L-His was purified by Ni-affinity column and used to immunize mice. The antiserum was collected after eighth weeks. The spleen cells from the immunized mice were fusion with SP2/O myeloma cells. The polyclonal antibody against GIV-5L and the monoclonal antibody against GIV-120L were produced.

The results of temporal expression pattern analysis and drug inhibition assay in GIV-infected GK (grouper kidney) cells showed GIV-5L and 120L as late genes. The transtripts of GIV-5L was detected at 9 to 30 h post-infection (p.i.) and the same result at protein level. The transtript of GIV-120L was detected at 12 to 30 h p.i. and the protein level was detected at 18 to 30 h p.i. The localization of GIV-5L was in the cytoplasm at 12 h p.i. and aggregated to the nuclear at 24 h p.i. The localization of GIV-120L was at virus assembly site at 24 h p.i. This study identified two late genes, GIV-5L and 120L. These results contribute to understanding of the development of GIV during virus assembly and application in immuno diagnostics on GIV.

中文摘要(Abstract in Chinese) I
英文摘要(Abstract in English) II
致謝詞(Acknowledgements) III
目錄(Table of Contents) IV
表目次(Table List) VI
圖目次(Figure List) VII
第一章、 前言(INTRODUCTION) 1
一、 石斑魚(Grouper) 1
二、 虹彩病毒(Iridovirus) 1
三、 石斑魚虹彩病毒(Grouper iridovirus) 5
四、 神經絲(Neurofilament, NF) 7
五、 實驗目的(Objective) 8
第二章、 實驗器材(MATERIALS) 9
一、 細胞株(Cell lines)、病毒(Virus)及菌種(Bacteria) 9
二、 原核表現載體(Bacterial Expression Vectors) 9
三、 耗材(Supplies) 9
四、 藥品(Chemical) 9
五、 儀器(Instrument) 12
第三章、 實驗方法(METHODS) 13
一、 基因序列分析(Sequence Analysis) 13
二、 細胞與病毒(Cell and Virus) 13
三、 表現載體構築(Construction of Expression Vector) 14
四、 重組蛋白製備(Preparation of Recombinant Protein) 16
五、 抗體製備(Preparation of Antibody) 19
六、 基因轉錄與轉譯分析(Transcriptome and Proteome Analysis) 21
第四章、 實驗結果(RESULTS) 24
一、 基因序列分析(Sequence Analysis) 24
二、 表現載體構築(Construction of Expression Vector) 25
三、 重組蛋白製備(Preparation of Recombinant Protein) 25
四、 抗體製備(Preparation of Antibody) 26
五、 基因轉錄與轉譯分析(Transcriptome and Proteome Analysis) 26
第五章、 討論(DISCUSSION) 27
參考文獻(References) 34
表與圖(Table and Figure) 43


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