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Virologica Sinica, 30 (5) : 379-385, 2015
Research Article
Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China
 Correspondence: yangkai@mail.sysu.edu.cn
(942.06KB)  
Abstract
In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome, and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6. Using the resulting ts helper plasmid pMON7124ts and the ts donor plasmid pFB1ts-PH-GFP, a recombinant bacmid, bAcWT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For bAcWT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.
Received: 25 Aug 2015  Accepted: 30 Sep 2015  Published online: 19 Oct 2015
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