A novel method for generating baculovirus bacmids using EGFP-mediated purification and linearization.

Abstract

Baculovirus bacmids have been widely used in over-expression and gene deletion. Traditionally, baculovirus bacmids are developed by inserting an 8.6 kbp bacterial DNA cassette into baculovirus genomes either through homologous recombination in cultured cells or via in vitro cloning. In this study, by introducing Bsu36i-attached egfp to the 8.6 kbp bacterial DNA cassette, we develop a novel method for generating baculovirus bacmids. An 11.6 kbp bacterial DNA cassette containing the introduced egfp was used to generate an intermediate bacmid. With the EGFP reporter, purification was performed in cultured cells, increasing the proportions of recombinants. The intermediate bacmid containing the 11.6 kbp bacterial DNA cassette was obtained by transforming DH10B competent cells with viral DNA after 3 rounds of purification. The intermediate bacmid DNA was linearized by digestion with Bsu36i and then was co-transfected with the PCR-amplified 8.6 kbp bacterial cassette into BmN cells, where homologous recombination occurred between them. The final BmNPV bacmid was obtained by transforming DH10B competent cells with viral DNA. Capable of increasing the proportions of recombinants via purification and linearization, this method has great potential to be used for bacmid generation for baculoviruses, especially those that are not capable of producing high titers of viruses.