Efficient Method to Generate Homologous Recombinant Baculovirus Genomes in E. coli

Here we describe a convenient method to generate homologous recombinant baculoviral genomes in E. coli. The recombination takes place with the aid of recombination enzymes provided by the phage lambda Red system between a bacmid (a baculoviral genome that can replicate in bacteria) and a linear fragment. Proof of concept was provided when the cathepsin gene (v-cath) of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) was replaced by the chloramphenicol resistance gene (CmR). First, CmR was inserted between the flanking sequences of the HaS-NPV v-cath. Each of the flanking regions was about 1 kb. The fragment was linearized and electroporated into bacteria containing both the HaSNPV bacmid and the lambda Red system. Recombinant bacmids resistant to chloramphenicol were selected. In comparison to the standard co-transfection/plaque assays, this method significantly reduces the time required to construct baculovirus knockout mutants. It may also be useful in the manipulation of other large viral genomes.