Pfastbac dual plasmid

According to previous studies of Mhp and PCV2, coding DNA sequences (CDS) of P97R1 (MHP168_110), P46 (MHP168_522), and P42 (MHP168_069) from Mhp strain 168 (GenBank CP002274.1) were used to construct the chimeric protein antigen; Cap protein (deleting the nuclear localization signal of 41 amino acid residues) from PCV2 isolate strain ShanDong3-2016 (KY656098.1) was used as another antigen (Table 1) [9 (link),15 (link),32 (link)]. EGFP and mCherry were used as tag proteins for detection and were cloned from vectors pEGFP-N1 and pmCherry-N1 (Clontech, Otsu, Shiga, Japan), respectively. SPgp64 (GenBank AFO10080.1) and TMDgp64 (GenBank CAA24524.1) were located upstream of the target protein and downstream of the tag protein, respectively.
Flexible linker GGSG (GlyGlySerGly) was inserted between each chimeric subunit protein of P97R1P46P42 to achieve proper folding. The fusion gene fragment SPgp64-P97R1P46P42-mCherry-TMDgp64-6×His was inserted into the baculovirus expression vector pFastBac dual plasmid (Invitrogen, Carlsbad, CA, USA) to generate pFastBac dual-P97R1P46P42-mCherry-Cap-EGFP. SPgp64-Cap-EGFP-TMDgp64-6×His was inserted into pFastBac dual to generate pFastBac dual-P97R1P46P42-Cap.

Production of MCPyV and TSPyV VLPs in insect cells has been described previously [15] (link), [28] (link). VLPs were also generated for PtvPyV1, PtvPyV2 and OraPyV1. Briefly, VP1 coding sequences were obtained by total synthesis with codon usage-adapted sequences for expression in Spodoptera frugiperda cells (Genscript, Piscataway, NJ, USA) (Sequences based on PtvPyV1b (HQ385747), PtvPyV2c (HQ385750) and OraPyV1 (FN356900)) [26] (link), [27] (link). The different VP1 genes were cloned under the control of the polyhedrin promoter of pFastBac Dual plasmid (Invitrogen, FisherScientific, Illkirch, France) and then used to generate recombinant baculoviruses using the Bac-to-Bac system (Invitrogen). HiFive cells maintained in Grace medium (Invitrogen) were infected with the different recombinant baculoviruses for production of the polyomavirus VLPs. VLPs were then purified by ultracentrifugation (18 h at 30,000 rpm in a Beckman SW 32 rotor) in a CsCl gradient and assembly of VP1 into VLPs was verified by electron microscopy. The preparations were applied to carbon grids, negatively stained with 1.5% uranyl acetate and observed with a JEOL 1011 electron microscope at 50,000 nominal magnification (Figure 2).