Puc57 simple plasmid

Chimaeric RHDV VLP were developed using a recombinant baculovirus expression system, as has been previously published [32 (link), 33 (link)]. In brief, the VP60 N-terminus for each construct was purchased as synthetic DNA in a pUC57-Simple plasmid (Genscript, New Jersey, USA). The N-terminus sequences were extracted and ligated with the remainder of RHDV VP60 in a pAcUW51 (GUS) expression plasmid. Expression plasmids were co-transfected into Spodoptera frugiperda (Sf21) cells grown in SF900-III medium (Invitrogen, Auckland, NZ) along with the FlashBAC ULTRA™ expression system (Oxford Expression Systems, Oxford, UK). Sequence identity was confirmed by sequencing (Massey Genome Service, NZ). Each recombinant baculovirus was plaque purified and amplified. VLP were expressed in Sf21 cells as has been previously published [33 (link)], including CsCl gradient purification by ultracentrifugation. Expressed recombinant VP60 constructs were confirmed through mass spectrometry using MALDI-TOF/TOF or a LTQ-Orbitrap hybrid (Centre for Protein Research, University of Otago, NZ).

RHDV VLP (VP60) and RHDV VLP engineered to express the tumour antigen epitope survivin_97-104 (Surv.VLP) were developed using a recombinant baculovirus expression system, as previously described [21 (link),22 (link)]. In brief, the DNA sequence encoding the survivin peptide was purchased as synthetic DNA in a pUC57-Simple plasmid (Genscript, Piscataway Township, NJ, USA). The survivin sequence was extracted and ligated with the RHDV VP60 in a pAcUW51 (GUS) expression plasmid. The expression plasmids (VP60 and Surv.VLP) were co-transfected with the FlashBAC ULTRA™ (Oxford Expression Systems, Oxford, UK) into Spodoptera frugiperda (Sf21) cells. To express VP60 and Surv.VLP, suspension cultures of Sf21 cells were infected with the respective recombinant baculovirus at a multiplicity of infection of 1 and incubated for 3 days at 27 °C with shaking at 125 rpm. To isolate VLPs, the cells were lysed with 0.5% Triton X-100, followed by differential centrifugation and a CsCl gradient using ultracentrifugation (100,000× g, 18 h, 4 °C). The VLP band was harvested and the VLP concentration determined by A280 absorbance by NanoDrop (Thermo Scientific, Rockford, IL, USA) using an extinction coefficient of 78,000 M⁻¹ cm⁻¹ and a molecular weight of 60,000 Da.