Rapid Disruption of Bombyx mori Nucleopolyhedrovirus orf60 by Red Recombination System

BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupt Bombyx mori nucleopolyhedrovirus (BmNPV) open reading frame 60 (orf60) in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113, which harbors plasmid pKD46 encoding λ Red recombinase, to produce E. coli BW25113-Bac. This could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with a length of 63 bp, which had 45bp homologous to the orf60 gene and 18 bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60 with the help of λ Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60 was not expressed in BmN cells infected with knockout bacmid.