王重雄臺灣大學:昆蟲學研究所陳韻如Chen, Yun-RuYun-RuChen2010-05-102018-06-292010-05-102018-06-292008U0001-2407200817254300http://ntur.lib.ntu.edu.tw//handle/246246/181745豆莢螟核多角體病毒 (Maruca vitrata multiple nucleopolyhedrovirus, MaviMNPV) 係分離自罹病之豆莢螟 [M. vitrata (Fabricius), Pyralidae]。目前豆莢螟核多角體病毒基因體已解序，其基因體大小約 111.9 kbp，共有 126 個 ORFs (open reading frames)。經由親緣關係分析證明其是屬於第一群核多角體病毒，與廣寄主域之 AcMNPV (Autographa californica MNPV, 加州苜蓿夜蛾核多角體病毒) 及 BmNPV (Bombyx mori NPV, 家蠶核多角體病毒) 最為相近；刪除非同源之基因，豆莢螟核多角體病毒開放譯讀區之排列和 AcMNPV 完全相同。本研究重點係以豆莢螟核多角體病毒在於非寄主細胞之感染試驗，基因組轉錄時序及表現載體構築為主軸，並進一步延伸至應用層面：發展新穎桿狀病毒之表現系統。作病毒感染試驗的細胞株包括 IPLB-LD-652Y (LD, 吉普賽舞蛾細胞株)、NTU-MV56 (豆莢螟細胞株)、NTU-LY4 (LY4, 黑角舞蛾細胞株)、NTU-PN-HH (HH, 榕樹透翅毒蛾細胞株) 及 Sf9 (秋行軍蟲細胞株) 細胞株。利用本研究構築的螢光病毒之感染試驗中，結果顯示僅有部分 LD 細胞可接受 MaviMNPV 感染，並且可產生具有感染力之病毒子代，感染後第七天病毒效價高達 105 TCID50 / ml。利用螢光訊號計算感染比例，與 MV 細胞可達到 99% 之感染率比較，僅有 LD (Lyamntria dispar) 細胞 (1%) 顯示出螢光訊號，其他感染之 Sf9、LY4 和 HH 細胞皆無病毒子代產生；利用及時定量 PCR 偵測 MaviMNPV 感染之 Sf9、LY4 及 HH 細胞感染試驗中，皆偵測不到病毒基因轉錄。此證據顯示 MaviMNPV 異於其他第一群鱗翅目 NPVs。利用微陣列晶片和即時定量 PCR 偵測 MaviMNPV 感染其寄主細胞後，病毒基因表現之時序分析，結果顯示出基因表現時序可區分為三期，早期表現之基因 (0-6 小時；0-6 hpi) 共 25 個 ORF，佔基因組之 19.84%；晚期表現之基因 (6-15 小時；6-15 hpi) 等共 75 個，佔基因組之 59.52%；非常晚期表現之基因 (15-48 小時；15-48 hpi) 共 26 個，佔基因組之 20.64%；偵測 MaviMNPV 感染的 LD 細胞中之 25 個 MaviMNPV ORFs 的表現，只有 14 個 ORFs 可偵測到，其中以 ie 家族和少數結構蛋白等有較穩定之表現。利用即時定量 PCR 比較多角體蛋白基因相對表現量，MaviMNPV/MV 細胞優於 AcMNPV/Sf 細胞，於是利用豆莢螟核多角體病毒之 polyhedrin 啟動子構築新穎傳送載體，pMV-polh 和 pMV-polh-DE，成功地表現出加強綠螢光蛋白 (eGFP)，並篩選出兩種轉綠螢光基因之 MaviMNPV 重組病毒，egfp-MaviMNPV1 和 egfp-MaviMNPV2，比較 MaviBEV1 或 2/NTU-MV 細胞、AcBEV (A. californica baculovirus expression vector)/Sf9 細胞，與 LyxyBEV (Lymantria xylina BEV)/LY 細胞之加強綠螢光蛋白產量，胞內以 AcBEV/Sf9 最高，而 MaviBEV2/NTU-MV 其蛋白質產量最低；而胞外螢光蛋白以 MaviBEV1/NTU-MV 細胞產量最高，而以 MaviBEV2/NTU-MV 產量最低。此結果顯示出 MaviBEV1/NTU-MV 細胞具有能媲美 AcBEV/Sf21 細胞表現系統之新桿狀病毒表現系統。總結目前的研究成果，MaviMNPV 不但具學術研究的價值，且深具應用於表現載體發展之潛能。A new baculovirus, Maruca vitrata multiple nucleopolyhedrovirus (MaviMNPV), is isolated from the moribund larvae. The whole MaviMNPV genome has been sequenced and analysed. The genome size is approximately 111.9 kbp, processing 126 open reading frames (ORFs). According to the phylogenetic analyses, MaviMNPV belongs to Group I NPV and is closely related to AcMNPV (Autographa californica MNPV) and BmNPV (Bombyx mori NPV). Comparing the localization of homologous ORFs, the ORFs arrangement of MaviMNPV is 100% matched to that of AcMNPV. The main objectives of this study were focused on the in vitro host range, MaviMNPV genes expression profile, and the transfer vector construction. Five cell lines, including IPLB-LD-652Y (LD), NTU-MV56, NTU-LY4 (LY4), NTU-PN-HH (HH), and Sf9, were chosen for MaviMNPV infection assays. Compared to 99% MV cells infected with egfp-MaviMNPV1, only 1% IPLB-LD-652Y cells were susceptible to MaviMNPV, and LD cells could produce virus progeny, and the virus titer could reach 105 TCID50 / ml; no virus progeny was produced in MaviMNPV-infected Sf9, LY4, and HH cells . The qPCR results also revealed that the genes of MaviMNPV could not be transcripted in the infected Sf9, LY4 and HH cells. These results supported that MaviMNPV is a distinct species of the group I lepidopteran NPVs. Based on the results of the microarray, the genes expression stages were divided into three groups: the early stage (0-6h), including 25 ORFs (19.84%); the late stage (6-15h), including 75 ORFs (59.52%); and the very late stage (15-48h), including 26 ORFs (20.64%). In the MaviMNPV-infected LD (Lymantria dispar) cells, 14 of the 25 ORFs were detectable. The ie family and several structure protein genes of MaviMNPV in LD cells showed the stable expression trend. The polyhedrin related expression levels was also detected by real-time qPCR. The results showed that MaviMNPV-infected MV cells could produce the greatest level of polyhedrin compared with that of AcMNPV-infected Sf cells. We had successively constructed two new transfer vectors, pMV-polh and pMV-polh-DE, driven by the MaviMNPV polyhedrin promoter. Two egfp recombinant MaviMNPVs, egfp-MaviMNPV1 and egfp-MaviMNPV2, were cloned. We compared the eGFP produced from intra- and extracellular levels of MaviBEV1/NTU-MV cells, MaviBEV2/NTU-MV cells, AcBEV (A. californica baculovirus expression vector, AcBEV)/Sf9 cell, and LyxyBEV (Lymantria xylina BEV, LyxyBEV)/LY cells. In the intracellular level, AcBEV/Sf9 cells produced the greatest level of eGFP while MaviBEV2/NTU-MV was the lowest BEV. In the extracellular level, MaviBEV1/NTU-MV cells was the highest production of eGFP while MaviBEV2/NTU-MV cells was the lowest BEV. Thererfore, MaviBEV1/NTU-MV has a high potential to be developed as a new BEVS for comparing with the commercial AcBEV/Sf9 cells. In conclusion, these results showed that MaviMNPV/NTU-MV cells possess a high potential not only for scientific studies but also BEVS application.目錄試委員會審定書......................................................................................................... i謝.................................................................................................................................. ii文摘要..........................................................................................................................iii文摘要...........................................................................................................................v一章、緒言.....................................................................................................................1二章、研究背景與前人研究.........................................................................................2.1 豆莢螟之介紹.............................................................................................................2.2 桿狀病毒之簡介.........................................................................................................2.3 桿狀病毒之分類.........................................................................................................3.4 桿狀病毒之生活史...................................................................................................4.5 桿狀病毒寄主域之研究...........................................................................................6.6 微陣列技術 (Microarray) 在桿狀病毒研究上之運用.............................................6.7 桿狀病毒表現載體系統之發展...............................................................................7.8 豆莢螟核多角體病毒基因體之之解序與研究.......................................................9三章、材料方法.........................................................................................................12.1 豆莢螟核多角體病毒超微形態之觀察...................................................................12.1.1 供試細胞株............................................................................................................12.1.2 供試病毒株............................................................................................................12.1.3 病毒超微結構觀察................................................................................................12.2 豆莢螟核多角體病毒體外寄主域之研究...............................................................13.2.1 供試細胞株............................................................................................................13.2.2 供試病毒株............................................................................................................13.2.3 病毒效價之定量 (TCID50)...................................................................................13.2.4 細胞感染試驗.......................................................................................................13.2.5 感染細胞 RNA 之萃取與 DNA 之去除...............................................................14.2.6 cDNA 之製備......................................................................................................14.2.7 real-time PCR 檢測.............................................................................................14.2.8 螢光病毒感染試驗...............................................................................................15.2.9 聚丙烯醯胺膠體電泳 (SDS-PAGE) ...............................................................15.2.9.1 膠體製備........................................................................................................15.2.9.2 樣本處理.........................................................................................................16.2.9.3 西方墨點法 (Western blot hybridization) ..........................................................16.3 豆莢螟核多角體病毒基因體表現平台之建立.....................................................16.3.1 病毒多角體之純化...............................................................................................16.3.2 病毒 DNA 之萃取...............................................................................................17.3.3 細胞 DNA 之萃取.................................................................................................17.3.4 基因之選殖...........................................................................................................18.3.5 轉型作用...............................................................................................................19.3.6 質體 DNA 之萃取.................................................................................................19 .3.7 限制酶切割反應.................................................................................................19.3.8 晶片置備............................................................................................................20.3.9 細胞感染試驗......................................................................................................20.3.10 RNA 萃取.............................................................................................................20.3.11 mRNA之純化...................................................................................................21.3.12 mRNA 標定 Cy5.................................................................................................21.3.13 純化標訂好之產物.............................................................................................21.3.14 RNA Fragmentation............................................................................................22.3.15 雜合反應............................................................................................................22.3.15.1 前雜合反應......................................................................................................22.3.15.2 雜合反應..........................................................................................................22.3.15.3 雜合後清洗步驟...............................................................................................23.3.15.4 最後之清洗.................................................................................................23.3.16 訊號掃描...................................................................................................23.3.17 Stripping 去雜合..........................................................................................24.3.18 晶片訊號殘留與否之確認............................................................................24.3.19 基因轉錄訊號之分析......................................................................................24.3.20 利用基因表現時序將基因分群....................................................................24.3.21 利用聚合酶連鎖反應 (PCR，polymerase chain reaction) 確認引子對之專一 .................................................................................................25.3.22 利用即時定量 PCR 檢測豆莢螟核多角體病毒之基因表現....................... 25.3.23 利用基因表現時序將基因分群.......................................................................... 26.3.24 MaviMNPV 感染 LD 細胞株試驗與病毒基因表現之偵測..............................26.4 豆莢螟核多角體病毒 / 豆莢螟細胞株表現系統之建立........................................26.4.1 供試細胞株.....................................................................................................26.4.2 供試病毒株....................................................................................................27.4.3 多角體蛋白啟動子序列預測與比對.................................................................27.4.4 病毒效價之定量 (TCID50) ..................................................................................27.4.5 細胞感染試驗....................................................................................................27.4.6 感染細胞 RNA 之萃取與 DNA 之去除................................................................28.4.7 cDNA 之製備....................................................................................................28.4.8 即時定量 PCR 檢測 polyhedrin 相對表現量.......................................................28.4.9 傳送載體之構築..................................................................................................28.4.9.1 前後同源序列增幅.............................................................................................28.4.9.1.1 聚合酶鏈鎖反應 (polymerase chains reaction, PCR) ……..............................28.4.9.1.2 PCR 產物之純化、限制酶處理與產物確認....................................................28.4.9.2 綠螢光蛋白基因之選殖...................................................................................29.4.10 轉染作用 (transfection)...................................................................................29.4.10.1 共轉染作用 (co-transfection) .......................................................................29 .4.10.2 傳送載體轉染與感染之病毒進行重組.......................................................30.4.11 轉基因病毒之單株化.....................................................................................30.4.12 病毒斑試驗 (Plaque assay) .............................................................................30.4.13 細胞感染試驗..................................................................................................30.4.14 細胞蛋白之收取..............................................................................................31.4.15 螢光蛋白相對值之測量 (Relative Fluorescent Unite, RFU).......................31.4.15.1 感染細胞之蛋白質取樣..................................................................................31.4.15.2 蛋白質定量....................................................................................................31.4.15.3 螢光值測量.......................................................................................................32四章、結果................................................................................................................33.1 豆莢螟核多角體病毒超微構造之觀察...............................................................33.2 豆莢螟核多角體病毒體外寄主域之研究..............................................................33.2.1 病毒感染試驗......................................................................................................31.2.2 及時定量 PCR 檢測...............................................................................................34.3 豆莢螟核多角體病毒基因體表現平台之建立..................................................34.3.1 MaviMNPV 在感染 MV 細胞株後基因體表現時序之建立................................34.3.2 MaviMNPV 感染 LD 細胞株試驗.........................................................................36.4 豆莢螟核多角體病毒 / 豆莢螟細胞株表現系統之建立.....................................37.4.1 預測之啟動子序列比較...............................................................................37 .4.2即時定量 PCR 檢測多角體蛋白基因相對表現量..............................................37.4.3 傳送載體之建構..................................................................................................38.4.4 轉綠螢光基因病毒之篩選.................................................................................38.4.5 螢光病毒時序性感染試驗 ..............................................................................38.4.6 利用螢綠螢光蛋白產量比較三種桿狀病毒表現載體之產量........................39五章、討論............................................................................................................40.1 豆莢螟核多角體病毒超微構造之觀察...................................................................40.2 豆莢螟核多角體病毒體外寄主域之研究.........................................................40.3 豆莢螟核多角體病毒基因體表現平台之建立................................................44.4 豆莢螟核多角體病毒 / 豆莢螟細胞株表現系統之建立....................................48六章 結論................................................................................................................53考文獻.........................................................................................................................54表.................................................................................................................................72圖.................................................................................................................................78錄...........................................................................................................................97錄一、豆莢螟核多角體病毒基因組圖譜..............................................................94錄二、豆莢螟核多角體病毒與其他桿狀病毒演化親緣關係之分析.......................95錄三、MaviMNPV 開放譯讀區之預測表.................................................................96 錄四、即時定量 PCR偵測 MaviMNPV 感染 NTU-MV56 細胞後基因表現時序...........................................................................................................101錄五、MaviMNPV 在 MaviMNPV 感染之 LD 細胞內基因表現之狀況................108錄六、pIRES-EGFP 載體圖...................................................................................109錄七、(a) pMV-polh載體圖 (b) multiple cloning site................................................110錄八、pMV-polh-DE 載體圖...................................................................................111錄九、本實驗中所使用的引子名稱與序列.............................................................112錄十、 豆莢螟核多角體病毒基因表現分析平台之探針設計................................114錄十一、 豆莢螟核多角體病毒基因表現分析之即時定量PCR引子對設計........125次一、感染 egfp-MaviMNPV 7 天後平均螢光細胞數目之計量.............................69二、感染 egfp-MaviMNPV 7、8、9 天後病毒效價之結果...............................70三、豆莢螟核多角體病毒之基因表現時序性分析..............................................71 次一、NTU-MV 細胞感染 MaviMNPV 5天後之超微結構...................................75二、MaviMNPV包埋體之超微結構...................................................................76三、 Sf9、NTU-PN-HH、NTU-LY、IPLB-LD-652Y、和 NTU-MV56 五株細胞感染 MaviMNPV 3 天候之細胞病徵觀察.....................................................77四、polh 啟動子在 egfp-MaviMNPV 感染 (A) 3天 (B) 7天時細胞內表現 eGFP 之觀察.............................................................................................................78五、利用西方墨點法比較 LD 細胞與 1/2 到 1/100 倍稀釋之 MV 細胞蛋白質在感染 egfp-MaviMNPV 於 3 天與 7 天後螢光蛋白之產量...............................79六、及時定量 PCR 偵測感染 MaviMNPV 於 72 與 168 後ie-1、vp39 和 polyhedrin 三個基因在 MaviMNPV 接受與不接受細胞株內轉錄之結果................80七、以 18S 為控制組計算 MaviMNPV 感染 MV56 細胞之基因時序性表現.............................................................................................................................82八、MaviMNPV 在 MaviMNPV 感染之 LD 細胞內基因表現之狀況........85九、AcMNPV、MaviMNPV、LyxyMNPV 預測啟動子序列之比較.....................................................................................................................87十、polyhedrin 在 MaviMNPV感染之 MV56 細胞及 AcMNPV 感染之 Sf9、Sf21-AE 感染後第3、4、5天時表現量之偵測.......................................................88十一、野生型 MaviMNPV 與 egfp-MaviMNPV 共感染 MV細胞株........................89十二、 egfp 重組 MaviMNPV 感染 NTU-MV 細胞形成之螢光細胞群落................90十三、eGFP 在 egfp-MaviMNPV 感染之 NTU-MV 細胞株中時序表現狀況.........91十四、利用相對螢光值 (Relative Fluorescent Unit, RFU) 比較egfp-MaviMNPV1/NTU-MV56 細胞、egfp-MaviMNPV2/NTU-MV56細胞egfp-AcMNPV/Sf9 細胞、egfp-LyxyMNPV/NTU-LY1 細胞與四種桿狀病毒表現系統螢光蛋白之產量.............................................................................................92application/pdf2449881 bytesapplication/pdfen-US豆莢螟核多角體病毒桿狀病毒感染試驗微陣列生物晶片桿狀病毒基因表現時序桿狀病毒表現載體Maruca vitrata nucleopolyhedrovirus (MaviMNPV)baculovirus infection assaymicroarraybaculovirus genes expression profilebaculovirus expression vector system (BEVS)thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181745/1/ntu-97-F93632015-1.pdf