Introduction to baculovirus expression system|zoonbio

baculovirus expression system

The baculovirus expression system takes advantage of several facts about polyhedrin protein:
(1) that it is expressed at very high levels in infected cells, constituting more than half of the total cellular protein late in the infectious cycle;
(2) that it is nonessential for infection and replication of the virus, meaning that the recombinant virus does not require any helper function;
(3) that viruses lacking the polyhedrin gene have a laque morphology that is distinct from that of viruses containing the gene. Recombinant baculoviruses are generated by replacing the polyhedrin gene with a foreign gene through homologous recombinainserted into the viral genome and the polyhedrin gene is replaced. Thus recombinant viruses lack the polyhedrin gene and in its place contain the inserted gene, whose expression is under the control of the polyhedrin promoter.

Homologous recombination between circular wild-type DNA and the recombinant plasmid DNA occurs at a low frequency (typically 0.1% to 0.5%). This limitation was recently overcome by the development of viruses having Bsu36I restriction sites positioned within an essential gene—Open Reading Frame (ORF) 1629—which is downstream of the AcMNPV polyhedrin gene—and also in the upstream ORF 603—in such a way that digestion releases a fragment containing a sequence necessary for virus growth (Kitts and Possee, 1993). When insect cells are cotransfected with an appropriate recombinant transfer plasmid and linearized ORF 1629–deleted baculovirus DNA, the necessary ORF 1629 is provided by the transfer plasmid through homologous recombination. The vast majority of the progeny viruses, in many cases >99.9%, that are derived from these cotransfections contain the repaired virus with the target gene, thus minimizing the need to screen and plaque-purify recombinants. Several companies market linearized ORF 1629–deleted AcMNPV DNA. To further facilitate the identification of recombinants, several of these commercially available baculovirus DNAs contain the bacterial lacZ gene, which codes for β-galactosidase in lieu of the AcMNPV polyhedrin gene, thereby allowing lacZ-negative recombinants to be distinguished visually from any residual nonrecombinant viruses via a plaque assay. Nonrecombinant viruses form blue plaques on Xgal plates because they contain a functional lacZ gene, whereas recombinants form colorless, opaque plaques. Recombinant viruses can also be identified by DNA hybridization and polymerase chain reaction (PCR) amplification.

Another rapid and efficient method for generating recombinant baculoviruses uses site-specific transposition to insert foreign genes by homologous recombination into a bacmid propagated in E. coli rather than in insect cells. In this case, recombinant viral DNA is isolated from individual bacterial colonies and is free of any wild-type viral DNA. Upon transfection of insect cells, recombinant virus is generated free of parental nonrecombinant virus, thereby eliminating the need for multiple rounds of plaque purification. This is the basis of the Bac-to-Bac mers (1992), Grabenhorst et al. (1993), James et al. (1995), Jarvis and Finn (1995), Davis and Wood (1995), and Ogonah et al. (1996).