Improving baculovirus recombination

Abstract

Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation.

Figure 1

Comparative maps of the baculovirus polyhedrin locus. (A) The polyhedrin locus and surrounding genes on the wild type AcMNPV (accession no. L22858) genome. (B) The intermediate viral derivative BacPak6 showing the position of Bsu36I sites introduced to allow genome linearisation prior to transfection (10). Note, Bsu36I sites present in the β-galactosidase gene are not shown. (C) The AcMNPV bacmid used for insertional mutagenesis of ORF1629.

Figure 2

Procedure for knockout of ORF1629. (A) The oligonucleotides used for knockout with salient features indicated. Solid underlining indicates sequence homology of oligonucleotides 1629 CM5 and 1629 BSU CM3 to AcMNPV nucleotides 5291–5339 and 5359–5413, respectively. Dotted underlining indicates homology to chloramphenicol acetyl transferase. The introduced Bsu36I site is indicated. (B) Summary of the steps involved in the ET cloning strategy resulting in creation of Bac10:KO₁₆₂₉.

Figure 3

Transfection dose response curve and characterisation of recombinants. (A) Bac10:KO₁₆₂₉ preparations were quantitated by OD₂₈₀ and introduced into Sf9 cells with saturating amounts (500 ng) of transfer vector using lipofectamine. Cells were harvested at 2 days post infection, resuspended in Facsflow (Beckton Dickenson) and the number of fluorescent cells determined. Data are number of GFP positive cells per 5000. Transfection with vector only gave no fluorescence and transfection by Bac10:KO₁₆₂₉ only gave no virus recovery. (B) Plaque assay of progeny recombinant baculoviruses expressing GFP. Three dishes with well isolated plaques were photographed following illumination by UV light transmitted by a UV/blue plate converter (UVP, Upland CA) and photographed through an orange filter. Examination of multiple dishes failed to reveal any non-fluorescent plaques. (C) GFP (arrowed) expression levels of four recombinant viruses (lanes 4–7) produced using modified bacmid DNA were compared with GFP expressed by a recombinant made using non-modified viral DNA (lane 2). Marker proteins with the molecular mass (in kDa) shown are in lane 1 and a non-infected cell extract in lane 3. No reduction in expression level was apparent between lanes 4–7 and lane 2.