發展 Bac-to-Bac 之桿狀病毒雙效表現載體

桿狀病毒的研究著重在昆蟲病毒的基本病毒學與生物技術之應用。桿狀病毒表達載體系統（BEVS）是以重組的昆蟲桿狀病毒感染昆蟲細胞來表現蛋白質，是分子生物學的一種重要工具。穿梭桿狀病毒表達載體可以發揮哺乳動物細胞與昆蟲細胞的功能, 讓他們的發展會得到一個重要的優勢。我們把內部核糖體進入位點（IRES）跟桿狀病毒基因組連結在一起，然後生成可同時表達兩種基因之雙順反子桿狀病毒表達載體。在這個報告中，我們試著建造一個桿狀病毒載體擁有使用Bac-to-Bac系統的依雙順反子表達載體之IRES。傳統方法使用在昆蟲細胞的同源重組, 不過Bac-to-Bac系統帶來比傳統方法更多的好處。Bac-to-Bac 質粒:　pFastBac1-Polh-MCS-Lir-DsRed2 已成功生成。這種IRES- 基於Bac-to-Bac　BEVS將促進快速和有效地產生重組桿狀病毒：節省時間表達桿狀病毒質粒，一步純化和擴增，多重組病毒的快與和同時隔離以及滴度測定。

關鍵字

桿狀病毒、桿狀病毒表達系統、Bac-to-Bac系統、重組蛋白

並列摘要

Baculoviruses are one of the most studies insect viruses both in basic virology research and in biotechnology applications. This insect viruses based baculovirus expression vector system (BEVS) is an important tool for molecular biology and widely used in insect cells. Development of a shuttle baculovirus expression vector which can function in both the mammalian cell as well as in insect cell would have a significant advantages. We had cooperated an internal ribosome entry site (IRES) in to the baculovirus genomes and generated a bicistronic baculoviruses expression vector that can express two genes simultaneously. In this project, we attempt to construct a baculovirus vector with an IRES based bicistronic expression vector which using Bac-to-Bac system. The Bac-to-Bac system bring more benefits than the traditional methods using homologous recombination in insect cells. Bac-to-Bac plasmids:　pFastBac1-Polh-MCS-Lir-DsRed2 had been successfully generated. Such IRES-based Bac-to-Bac BEVS will facilitate rapid and efficient generation of recombinant baculoviruses: time saving expression bacmid, one-step purification and amplification, rapid and simultaneous isolation of multiple recombinant viruses as well as titer determination.

並列關鍵字

Baculovirus, Baculovirus expression system, Bac-to-Bac system, recombinant protein.

參考文獻

Basil MA. 2005. A brief journey with insect viruses with emphasis on baculoviruses. Journal of Invertebrate Pathology .89: 39-45.

D.R.OReilly, L.K.Miller, V.A.Luckow. 1992.Baculovirus Expression Vector: A Laboratory Manual, W.H. Freeman, NY.

T.S. Gritsun,M.V. Mikhailov,P. Roy,E.A. Gould. 1997. A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses.Nucleic Acids Res. 25 1864-1865.

G. Patel, K. Nasmyth, N. Jones. 1992. A new method for the isolation of recombinant baculovirus. Nucleic Acids Res. 20:97-104.

Cheng, T., C.Y. Xu, Y.B. Wang, M. Chen, T. Wu, J. Zhang, and N.S. Xia. 2004. A rapid and efficient method to express target genes in mammalian cell by baculovirus. World journal of gastroenterology: WJG. 10:1612-1618.

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發展 Bac-to-Bac 之桿狀病毒雙效表現載體

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