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研究生:周宗錄
研究生(外文):Tsung-Lu Chou
論文名稱:蝦白點症病毒結構性蛋白VP51A(ORF294)特性分析
論文名稱(外文):Characterization of Shrimp White Spot Syndrome Virus (WSSV) Structural Protein VP51A (ORF294)
指導教授:張雲祥張雲祥引用關係
指導教授(外文):Yun-Shiang Chang
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:68
中文關鍵詞:白點症病毒結構性蛋白質外套膜蛋白質
外文關鍵詞:WSSVstructural proteinenvelope protein
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白點症病毒 (White spot syndrome virus, WSSV) 是一個重要的甲殼類病毒,且會造成養殖蝦類的大量死亡。蝦白點病毒本身具有約300 kbp的基因體,是一個大型的雙股DNA病毒。目前以有58個病毒的結構性蛋白質被鑑定出來,本研究主要針對其中一個由白點症病毒ORF294 (GeneBank accession no. AF440570) 所轉譯出之結構性蛋白質VP51A作進一步的探討。由快速擴增cDNA 5’/3’端基因結構分析顯示,vp51A的主要轉錄起點位於ATG上游135 bp;不具有TATA box或類似的保守性序列;加聚腺嘌呤訊號恰位於基因轉譯停止密碼TAA重疊,而加聚腺嘌呤位置則位於加聚腺嘌呤訊號下游22 bp。此vp51A在病毒感染後6小時開始進行轉錄表現,且表現量會隨著感染時間而增加。電腦程式預測指出,VP51A譯讀區第37到43胺基酸呈現一個入核訊號 (nuclear localization signal, NLS) 的保守性序列,但於Sf9細胞中進行的實驗並未證實此一預測。純化病毒顆粒的免疫電顯分析以及配合鹽度梯度處理和超高速離心分離完整病毒顆粒組成後之西方轉漬雜合反應顯示,VP51A屬於病毒envelope蛋白質。另外,VP51A的西方轉漬雜合分析的結果也發現,除了預期的53 kDa的片段大小外,尚可偵測到72 kDa以及其他小分子量蛋白質出現;相似的結果也出現在病毒感染蝦組織及昆蟲細胞表現的重組VP51A上但利用細胞外轉錄轉譯系統生產的VP51A則僅出現一條72 kDa的蛋白質;上述結果顯示VP51A可能先表現出一個大分子量的蛋白 (72 kDa) 後再經過切割形成53 kDa及其他較小分子量的蛋白質。而其他藉由蛋白質切位突變實驗及以衍生自VP51A譯讀區不同區域片段抗體的西方轉漬雜合分析顯示,其大部分切位可能位於靠近N端之區域,然而,這些不同大小的VP51A蛋白質的生理意義為何,仍須進一步探討。
White spot syndrome virus (WSSV) is an important crustacean virus causing high mortality in cultured shrimp. WSSV is a double-stranded DNA virus with a genome size of about 300 kbp. So far, 58 viral structural proteins were identified. In this research, one of the structural protein translated form ORF294 (GeneBank accession no. AF440570), the VP51A, was studied. Gene structure analysis showed that the transcription initiation site of vp51A was located 135 bp upstream of the translation start codon ATG. TATA box, or its related consensus sequence was not recognized is 5’ untranslated region of this gene. The poly-A addition signal was overlapped with the translation stop codon TAA and the poly-A tail was added 23 bp downstream of the stop codon.The vp51A transcripts was observed 6 hours after virus infection and the expression levels increasing with the infection time course. Computer software anlysis discovered a conserved sequence of the nuclear localization signal (NLS) between 37 and 43 of VP51A coding region, but such prediction wasn’t confirmed by the following in vitro analysis performed in Sf9 cells. Immunoelectron microscopy analysis and Western blot hybridization performed on intact virus particle and separated viral components showed that the VP51A is an envelope protein. Furthermore, Western blot analysis of WSSV virion also demonstrated that except the expected 53 kDa band, there were another protein bands such as an obvious signal around 72 kDa and some other small molecular weight proteins exist. Similar result was found in the Western blot results performed on WSSV infect shrimp tissues and recombinant VP51A expressed insect cells. But when using the in vitro transcription and translation system to express the recombinant VP51A it demonstrated a 72 kDa protein only. This result showed that the VP51A gene might expresses a large molecular weight protein first and it will then be processed into another lower molecular weight ones. Other experiments, including the predict protein cutting site mutation of VP51A and Western blot hybridization by VP51A different region fragments derived antibadies suggested that most of the cutting sites of VP51A might distribute closer to the N-terminal region. The is processed and what are the biological meanings of there different types of VP51A proteins, still left to be elocudate.
封面內頁
簽名頁
授權書………………………………………………………...........…iii
中文摘要………………………………………………………….......iv
英文摘要……………………………………………………….......…vi
誌謝…………………………………………………………….....…viii
目錄…………………………………………………………….....…...x
圖目錄…………………………………………………………….....xiii
表目錄………………………………………………………….....….xv

1. 前言…………………………………………………………….......1
1.1 白點症病毒……………………………………………….…..1
1.2 結構性蛋白質之重要性………………………………….…..5
1.3 白點症病毒結構性蛋白質之發現與應用…………....……...5
1.4 研究目的……………………………………………………...7
2. 材料方法……………………………………………………….......9
2.1 基因結構分析…………………………………………...…....9
2.1.1 樣品來源………………………………………...….....9
2.1.2 total RNA萃取…………………………………..........9
2.1.3 反轉錄聚合酶鏈反應(reverse transcription polymerase chain reaction, RT-PCR)………..….....…....................10
2.1.4 基因表現時序分析………………………………..…11
2.1.5 快速擴增cDNA 5’/3’端…………………………......11
2.2 蛋白質表現及特性分析………………………………….....14
2.2.1 VP51A之細胞內表現位置分析…………………...…14
2.2.2 抗體製備………………………………………...........16
2.2.3 VP51A於病毒顆粒之定位分析…………………...…18
2.2.3.1 白點症病毒純化…………….………….........18
2.2.3.2 白點症病毒之抗VP51A C端抗體西方轉漬分析………………………..……………....……19
2.2.3.3 以西方轉漬分析VP51A於白點症病毒顆粒上之定位. ……..……………………………..…20
2.2.3.4 以免疫電顯分析VP51A於白點症病毒顆粒上之定位..………………..…………………......20
2.2.4 VP51A分子量變異之分析..........................................21
2.2.4.1 VP51A試管內轉錄及轉譯 (Transcription/ TranslationSystems, TNT)分析………….…..21
2.2.4.2 昆蟲細胞Sf9表現重組VP51A之抗V5抗體西方轉漬分析……………………………..............22
2.2.4.3 白點症病毒顆粒、病毒感染蝦組織及昆蟲細胞Sf9抗VP51A C端抗體西方轉漬分析..........23
2.2.4.4 白點症病毒顆粒、病毒感染蝦組織及昆蟲細胞Sf9抗VP51A中段片段抗體西方轉漬分析………………………………………..........23
2.2.4.5 VP51A切割位分析………………………..…24
3. 結果與討論…………………………………………………….…25
3.1 基因結構分析……………………………………………….25
3.1.1 基因表現時序分析…………………………………..25
3.1.2 快速擴增cDNA 5’/3’端……………………………..26
3.2 蛋白質表現及特性分析…………………………………….26
3.2.1 VP51A之細胞內表現位置分析…………………..…26
3.2.2 VP51A於病毒顆粒之定位分析…………………..…27
3.2.3 VP51A分子量變異之分析…………………………...28
3.2.3.1 VP51A試管內轉錄及轉譯分析………….….28
3.2.3.2 昆蟲細胞Sf9表現重組VP51A之抗V5抗體西方轉漬分析………………………….……....29
3.2.3.3 白點症病毒顆粒、病毒感染蝦組織及昆蟲細胞Sf9抗VP51A C端抗體西方轉漬分析…….29
3.2.3.4 白點症病毒顆粒、病毒感染蝦組織及昆蟲細胞Sf9抗VP51A中段片段抗體西方轉漬分析…………………………………………….30
3.2.3.5 VP51A切割位分析…………………………..30
4. 結論.………………………………………………………………31
參考文獻……………………………………………………………..59
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