- 學位論文
豆莢螟核多角體病毒基因體之研究與表現載體之構築
Genomic study and BEVS construction of Maruca vitrata multiple nucleopolyhedrovirus, MaviMNPV
摘要
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。
並列摘要
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.