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研究生:朱彥昌
研究生(外文):YAN-CHANG ZHU
論文名稱:藉由共感染不同之桿狀病毒來探討桿狀病毒宿主域的研究
論文名稱(外文):Investigating baculovirus’s host range through co-infect different baculoviruses
指導教授:吳宗遠
指導教授(外文):Tzong Yuan Wu
學位類別:碩士
校院名稱:中原大學
系所名稱:生物科技研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:60
中文關鍵詞:MaviMNPVAcMNPV共感染螢光報導基因BmNPV
外文關鍵詞:fluorescent expressionCo-infectAcMNPVMaviMNPVBmNPV
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桿狀病毒依其分離自何宿主而命名,大多數桿狀病毒擁有專一宿主域,但有部分桿狀病毒具有廣泛的宿主域。例如加州苜蓿夜蛾核多角體病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV),可感染33種鱗翅目昆蟲與多種昆蟲細胞株。與AcMNPV親源相似的有豆筴螟核多角體病毒(Marca vitrata multiple nucleopolyhedrovirus, MaviMNPV) 與家蠶核多角體病毒 (Bombyx mori multiple nucelopolyhedrovirus, BmNPV)。AcMNPV、BmNPV、MaviMNPV各自可感染Sf 21、BmN、NTU-MV532 細胞株。雖然三株病毒十分相似,卻不能感染另兩種非接受性細胞株。本研究使用帶有螢光基因的AcMNPV,BmNPV,MaviMNPV桿狀病毒分別對Sf21、BmN、NTU-MV532細胞株進行共感染,藉此探討病毒與病毒,病毒與宿主之間的交互作用。同時利用AcMNPV與MaviMNPV交叉感染Sf 21與NTU-MV532細胞株,期望能獲得單株具有感染Sf 21與NTU-MV532能力的跨宿主重組病毒。另一方面,我們於Sf 21細胞株先感染MaviMNPV 2,6,12,48小時後再感染AcMNPV,AcMNPV有協助MaviMNPV於Sf 21細胞中的複製,同時MaviMNPV亦有延遲AcMNPV的複製情形;當在NTU-MV532細胞株先感染AcMNPV 2,6,12,48小時再感染MaviMNPV,MaviMNPV反有拮抗作用,同時AcMNPV抑制MaviMNPV的複製。而在Sf 21細胞株中先感染BmNPV 2,6,12,48小時再感染AcMNPV,AcMNPV協助BmNPV
的複製,同時AcMNPV的複製受到BmNPV呈現延遲的結果。在BmN細胞中先感染AcMNPV 2,6,12,48小時再感染BmNPV有互相拮抗複製的結果。我們利用即時定量PCR來偵測各時期的表現。在Sf 21共感染MaviMNPV與AcMNPV,共感染下的AcMNPV的IE-1,IE-2,lef-2 36小時前都較單獨感染的高,而共感染下的MaviMNPV病毒基因表現則是高於單獨感染;在NTU-MV532中共感染AcMNPV與MaviMNPV,AcMNPV與MaviMNPV 的IE-1,IE-2,lef 2在共感染的情況下幾乎沒有表現。而為避免共感染克服宿主域是因為外泌性的因子影響,我們使用超高速離心機離心被AcMNPV之病毒上清液製作成控制培養液再共培養被MaviMNPV感染之Sf 21細胞株,MaviMNPV的感染情形並不如與AcMNPV共感染良好,從而屏除了外泌性因子的因素。

Baculovirus is named after the host that it infects. Most of baculoviruses have their own specific host range, but some have wide host range that can infect many different insectspecies, like Auotographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect 33 kind of insects and different kind of insect cell lines. Moreover, Maruca vitrata multiple nucleopolyhedrovirus (MaviMNPV) and Bombyx mori multiple nucleopolyhedrovirus (BmNPV) are two baculovirus that are similar in genome and structure. Despite the similarity in genome and structure, they can only infect their host range for instance AcMNPV infects sf21 cell line, BmNPV infects BmN cell line, and MaviMNPV infects NTU-MV532 cell line. We tried to find out the relationship between viruses and host their interaction. Different baculoviruses was incorporated with a different fluorescent reporter gene to co-infect Sf21,BmN and NTU-MV532 cell lines. Recombinant virus of AcMNPV and MaviMNPV was produced by cross infecting in Sf21 and NTU-MV532 which could infect both Sf21 and NTU-MV532 cell lines.

Late gene expression of respective gene driven by polyhedrin promoter was studied by observing their fluorescent expression after single or co-infection with AcMNPV and MaviMNPV in Sf21 and NTU-MV532 cells lines at different time intervals. Co-infection of AcMNPV and MaviMNPV in Sf21 cell suggests that AcMNPV and MaviMNPV help each other’s gene expression in late phase, but blocks gene expression in NTU-MV532 cell line. Similar, result was observed in BmN and Sf21 cells which was co-infected with AcMNPV and BmNPV. Additionally, to study the influence of secretion factor in host range ,condition medium was prepared byultracentrifugation of medium infected with AcMNPV which was used to culture theMaviMNPV infected Sf21 cells.The result suggests that the secretion factor didn't play any role in influencing host range.QRT-PCR was performed to identify the genes that are essential for specific host range and several early and core genes were targeted for the study and their gene expression was measured in different time intervals.The expression of IE-1, IE-2 and lef-2 geneswere higher in co-infectedSf21cells with AcMNPV and MaviMNPV at 24 and 36 hpi; than single infection of AcMNPV. However, the expression of IE-1, IE-2 and lef-2 geneswas higher in co-infected Sf21 cells with AcMNPV and MaviMNPV than single infection of MaviMNPV. No gene expression of IE-1, IE-2 and lef-2 genes observed inNTU-MV532 cells co-infected with AcMNPV and MaviMNPV or AcMNPV alone but high expression of genes observed in NTU-MV532 cells infected with MaviMNPV alone.

摘要 I
Abstract III
目錄 V
圖目錄 VIII
背景介紹與文獻回顧 1
1.1桿狀病毒簡介 1
1.1.2桿狀病毒於農業上應用 2
1.2.1桿狀病毒宿主域 3
1.2.2 DNA helicase 在BmNPV與AcMNPV對宿主域的影響 3
1.2.3 使用 polyhedron promoter 主導的報導基因觀察宿主域 4
1.2.4 IE-1與hrs交互作用對宿主域的影響 4
1.2.5晚期基因表現狀況對宿主域的影響 5
1.3.1豆莢螟核多角體病毒(MaviMNPV) 5
1.4.1螢光報導基因 6
1.4.2綠螢光蛋白 Green fluorescent proteins (GFP) 6
1.4.3 紅螢光蛋白 7
材料與方法 8
2.1材料 8
2.1.1藥品與試劑 8
2.1.2 儀器設備 9
2.1.3昆蟲細胞株 9
2.2實驗方法 10
2.2.1 限制酶剪切 10
2.2.2 DNA agarose 膠體電泳 (gel electrophoesis ) 10
2.2.3 DNA 片段的回收 (Elution ) 11
2.2.4 DNA 黏合 (Ligation ) 12
2.2.5 聚合酶連鎖反應 (polymerase chain reaction,PCR ) 12
2.2.6質體的轉型 (Transformation ) 13
2.2.7質體的純化 13
2.2.8冷凍細胞株 14
2.2.9解凍細胞株 15
2.2.10病毒感染 15
2.2.11 RNA 萃取 15
2.2.12cDNA製備 16
2.2.13即時定量PCR 17
結果: 18
3.1 Mini-AcMavi 重組病毒挑選純化 18
3.2不同時間點感染不同病毒對晚期基因表現的影響 19
3.3 病毒之間的交互作用與晚期基因表現時程比較 21
3.4 共感染下的即時基因表現 22
3.5使用控制培養液排除外泌性因子的影響 24
討論: 25
4.1 宿主域的擴展與細胞接受性 25
4.2 病毒之間的交互作用影響晚期基因表現 26
4.3 不同時期病毒基因表現會受外來病毒影響 28
4.4 宿主域的擴展並非藉由外泌性因子的影響 29
參考文獻: 48
圖目錄
圖 1 33
圖 2 34
圖 3 35
圖 4 36
圖 5 38
圖 6 40
圖 7 42
圖 8 45
圖 9 46
圖 10 47
圖 11 50



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