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  • 學位論文

於桿狀病毒表現系統中以RhPV 5’UTR IRES同時表現不同胞器標的蛋白特性分析之研究

Analysis of simultaneously production of different organelles targeting proteins by RhPV 5’UTR IRES in Baculovirus expression system

指導教授 : 吳宗遠
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摘要


於桿狀病毒昆蟲表現系統中,利用RhPV 5’ UTR IRES構築出一個可轉譯雙基因的表現載體。重組桿狀病毒中包含了紅螢光 (DsRed)及綠螢光基因 (EGFP),其基因分別位在RhPV 5’ UTR IRES的上下游區。紅螢光基因藉由Cap-dependent translation之機制所轉譯;同時地,綠螢光基因由IRES-dependent進行之後的轉譯,兩者轉譯機制不同。將此所選殖出的Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 重組病毒感染於Spodoptera frugiperda 21 cells (SF21)時,可在晚期強勢啟動子(polyhedrin promoter)驅動下同時生產出兩種蛋白質。為了要更進一步拓展此雙效表現載體的應用性,我們利用此方法結合可與胞器專一性標定的螢光蛋白,發展出一套可以同時標定不同胞器之技術。由先前文獻中 (Tao et al.,1997) 得知,AcMNPV之膜蛋白 ODV-E66的N端前23個胺基酸,能有效率的將蛋白質運輸至細胞核內膜上。我們將此23個胺基酸融合至紅螢光基因,使其做為標定細胞核的紅螢光蛋白。另外我們也發現將分泌性蛋白與綠螢光蛋白融合在一起時,綠螢光可拘留在細胞膜上,此可做為標定細胞膜的的綠螢光蛋白。此些重組病毒分別命名為 vAcNLSDRhirE 及vAcDRhirS-E等。藉由IRES可同時轉譯兩種蛋白質的特性,我們成功的標定出細胞膜,細胞核與細胞質等區域。證明細胞核與細胞質可同時地被不同的螢光蛋白所標定,利用此生物螢光標定不同胞器的技術可取代以往用化學藥劑或是抗體進行染色的方式。除此之外,也可以利用此技術觀察到細胞內的生物活動。結果證明,確實可以有效的利用RhPV 5’UTR IRES同時表現不同胞器之標的蛋白。

關鍵字

桿狀病毒 IRES 螢光蛋白 報導基因

並列摘要


A bi-cistronic baculovirus transfer vector was constructed based on the 5’UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene (DsRed) and green fluorescent protein gene (EGFP) flanking the RhPV 5’UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. To explore the application of the bi-cistronic expression vector, we employed this approach combined with the organelle targeting specific fluorescent proteins to develop a dual organelles labeling technique. For the N-terminal 23 amino acids of envelop protein ODV-E66 of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is sufficient to traffic fusion protein to intranuclear membranes, we fused the nuclear traffic signal peptides of ODV-E66 to Dsred2 as a first cistron (translated by CAP-dependent initiation) and EGFP as the second cistron (translated by RhPV IRES -dependent initiation) in the bi-cistronic baculovirus transfer vector and the resulting recombinant virus was named vAcNLD-ir-E. In vAcNLD-ir-E infected Sf21 cells, the red fluorescence was revealed on the nuclear membrane or within nuclear and the green fluorescence was full of the whole cell under fluorescent microscope. These results demonstrated that the nucleus and cytosol can be labeled with different fluorescence marker simultaneously by this novel bi-cistronic expression vector combined with the organelles targeting specific fluorescent proteins technique.

並列關鍵字

reporter gene baculovirus IRES fluorescent protein

參考文獻


Ellenberg Jan.,Jennifer Lippincott-Schwartz and John F. Presley. (1998) Two-Color Green Fluorescent Protein Time-Lapse Imaging. Biotechniques. 25,838-846.
Adam, S.A. (1999). Transport pathways of macromolecules between the nucleus and the cytoplasm. Curr. Opin. Cell Biol. 11, 402-406.
Blissard, G. W. and G. F. Rohrmann (1990) Baculovirus diversity and molecular biology. Annu. Rev. Entomol. 35: 127-55.
Braunagel, S.C., S.T. Willianson., S. Sakena., Z. Zhong., W.K. Russell ., M.D. Summers. (2004). Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: Facilitated trafficking to the inner nuclear membrane. PNAS. 101, 8372-8377.
Cole, N.B. (1998). Retrograde Transport from the Golgi to the ER. Cell boil. 140, 1-15.

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