Improved generation of recombinant baculovirus genomes in Escherichia coli

Abstract

An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of approximately 10(7)/microg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses. The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli.

Figure 1

Plasmid map of the pBVboost donor vector. The insect cell expression cassette is composed of a multiple cloning site (MCS, unique restriction enzymes shown) flanked by the polyhedrin promoter (pPolh) and simian virus 40 polyadenylation site (SV40 pA). Tn7L and Tn7R, left and right ends of the Tn7 cassette; SacB#3, mutated levansucrase gene; ori, the ColE1 origin of replication; GENT, gentamycin gene.

Figure 2

Verification of the transposition in the white colonies generated by the new pBVboost-based method. PCR analysis of (A) DH10Bac or (B) DH10BacΔTn7 cells transformed by pBVboost. A ∼2300 bp sized band was detected from the 10 randomly picked white colonies (lanes 1–10) indicating the presence of recombinant bacmids. A 325 bp band indicates the presence of parental bacmid. Lanes M, Ladder Mix molecular weight marker (Fermentas AB, Vilnius, Lithuania). Lane +, positive control (blue colony). Lane –, negative control (no template).

Figure 3

Overview of the BVboost baculovirus generation system.