The gene fragment encoding PVY CP was cloned from a complementary DNA (cDNA) of PVY-NTN strain (GenBank accession no. KM396648) and inserted into the vector pT7-7 (Addgene). The vector was used to transform Escherichia coli BL21(DE3) cells, which were grown to an optical density at 600 nm of 0.8 to 1.0 in 2× YT medium [tryptone (16 g/liter) (BD Biosciences), yeast extract (10 g/liter) (BD Biosciences), and NaCl (5 g/liter)] supplemented with 5 mM MgCl2 and 2 mM CaCl2. Protein expression was induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside for 20 hours at 20°C. Cells were lysed by sonication in phosphate-buffered saline (PBS) buffer [1.8 mM KH2PO4, 10.1 mM Na2HPO4 (pH 7.4), 140 mM NaCl, and 2.7 mM KCl], and cell debris was removed by centrifugation. The lysate was incubated for 20 min in a mixture of 4% PEG 8000 (Sigma-Aldrich) and 0.5 M NaCl to precipitate VLPs. The VLP suspension was centrifuged for 20 min at 14,000g, and the pellet was resuspended in PBS buffer by gentle overnight shaking. Remaining solid material was removed by 20 min of centrifugation at 35,000g. The supernatant was loaded on 20 to 60% sucrose density gradient and ultracentrifuged at 117,000g for 6 hours in a Beckman 50 Ti rotor. All fractions of the gradient were collected and analyzed with SDS–polyacrylamide gel electrophoresis (SDS-PAGE) to identify fractions containing VLPs. Selected fractions were pooled, dialyzed for 24 hours against PBS buffer, and concentrated using Amicon Ultra centrifugal filters with a 100,000 molecular weight cutoff (Millipore) to the final concentration of 1 to 7 mg/ml. All steps of the purification procedure including VLP storage were done at 4°C.

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