Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

• Published in: Virologica Sinica Vol. 30; no. 5; pp. 379 - 385
• Main Authors: Huang, Zhihong, Li, Ao, Pan, Mengjia, Wu, Wenbi, Yuan, Meijin, Yang, Kai
• Format: Journal Article
• Language: English
• Published: Wuhan Wuhan Institute of Virology, CAS 01-10-2015
• Subjects: Animals; Baculoviridae - genetics; baculovirus; baculovirus;Bac-to-Bac; Base Sequence; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell Line; Cloning, Molecular; DNA, Recombinant - genetics; Escherichia coli; Escherichia coli - genetics; expression; genes; genetic vectors; Genetic Vectors - genetics; Genome, Viral; heterologous gene expression; insects; Medical Microbiology; Microbial Genetics and Genomics; Microbiology; Oncology; plasmids; Plasmids - genetics; progeny; protein synthesis; Recombination, Genetic; replication origin; Research Article; Spodoptera - virology; syst; Temperature; Transfection; transposition (genetics); viral load; virion; Virology; temperature-sensitive plasmid; baculovirus; Bac-to-Bac baculovirus expression system; Tn7 transposition
• ISSN: 1674-0769; 1995-820X
• DOI: 10.1007/s12250-015-3638-8

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, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-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.