Improved baculovirus expression vectors

In recent years, the baculovirus expression vector system has become a well-established and popular method for producing high yields of structurally, functionally and antigenically authentic foreign proteins in insect cells. Traditionally, when making a recombinant baculovirus, the foreign gene is cloned into a transfer vector, which contains sequences that flank the polyhedrin (polh) locus in the virus genome. The virus genome (generally, Autographa californica multinucleopolyhedrovirus (AcMNPV)) and the transfer vector are introduced into the host insect cell, and homologous recombination between the flanking sequences common to both DNA molecules effects insertion of the foreign gene into the virus genome, resulting in a recombinant virus genome. The genome then replicates to produce recombinant budded virus (BV) that can be harvested from the culture medium. A major disadvantage of this method is that it generates a mixture of recombinant and original parental virus after the initial round of replication. Consequently, the recombinant virus has to be separated from the parental virus by plaque purification, a process that is labor-intensive, technically demanding and time-consuming. More recently, bacterial artificial chromosomes (BACs) have been incorporated into the baculovirus genome to allow recombinant virus construction in Escherichia coli. Several commercial bacmid-based systems are now available (e.g. Bac-to-Bac and BaculoDirect; Invitrogen), but all still produce a mixed pool of both parental and recombinant viruses, which then need to be separated from each other by antibiotic selection or nucleoside analogs.