Bac to bactm baculovirus expression system

Codon-optimized target genes were amplified by PCR using primers with appropriate restriction enzymes (Table 1), and the PCR products were inserted into the pFastBac^TM Dual vector. In brief, the pFB-chIL-12 plasmid contained chIL-12 under the control of the polyhedron (PH) promoter, and the EGFP gene, amplified from pcDNA™3.1 vector (Invitrogen, USA), was cloned downstream of the p10 promoter as a selection marker. The pFB-VP1/VP2 plasmid, which contained CAV VP1 and VP2 genes at independently separated expression cassettes driven by the PH promoters and the EGFP gene, was inserted in the vector, as described above (Figure 1). The recombinant plasmids were verified by sequencing, and transformed into MAX Efficiency^® DH10BAC^TM competent cells (Thermo, USA) to produce bacmids. The recombinant baculoviruses, B-chIL-12 and B-VP1/-VP2, were generated using the Bac-to-Bac^TM Baculovirus Expression System (Invitrogen, USA), following the manufacturer’s directions. Sf9 cells were used to generate recombinant baculoviruses and protein production via culture in a Sf-900^TM II media (Invitrogen, USA) at 27 °C.

Larvae of Spodoptera exigua were obtained from a healthy laboratory colony maintained on a semi-synthetic diet [23 (link)] at 25 ± 1 °C. Sf9 cells (ThermoFisher Scientific, Waltham, MA, USA) were maintained in TC100 medium (Lonza Bioscience, Cologne, Germany) supplemented with 10% fetal calf serum (Lonza Bioscience, Cologne, Germany) at 28 ± 1 °C [24 ]. For the construction of the recombinant virus expressing the enhancin gene, the Bac-to-Bac^TM Baculovirus Expression System was used (ThermoFisher Scientific, Waltham, MA, USA). The enhancin gene was amplified from Trichoplusia ni granulovirus (TnGV) [16 (link)], whereas the polyhedrin gene was amplified from the AcMNPV C6 clone, the type species of the Alphabaculovirus genus [25 (link)]. All viruses were obtained from the virus collection of the Microbial Bioinsecticides group at the Universidad Pública de Navarra.