Mosaic CuMVTT-RBM vaccine production, purification and analysis
For expression and purification of mosaic CuMVTT-RBM,E. coli C2566 (New England Biolabs, USA) competent cells were transformed with the plasmid pETDu-CMVB3d-nCoV-M-CMVtt.
After selection of clones with the highest expression level of target proteins, E. coli culture was grown in 100 ml of 2TY medium (1.6% trypton, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg/l) on an orbital shaker at 30°C to the OD(600) value of 0.8–1.0. Then, the cells were induced with 0.2mM IPTG, and the medium was supplemented with 5mM MgCl2. Incubation was continued on the rotary shaker (200 rpm, 20°C, 18h). The resulting biomass was collected by low-speed centrifugation and was frozen at -20°C. To disrupt the cells, the biomass was resuspended 10 ml of buffer (20mM Tris, 5mM EDTA, 5mM Et-SH, 5% glycerol, 10% sucrose, pH 8.0) and further treated with ultrasound (Hielscher 200, power 70%, pulse 50%, 16min) on ice. Then, 0.5% TX-100 was added and the solution was rotated at 10 rpm ON at 4°C without centrifugation. The solution was then clarified for 10min at 10000 rpm (Epperndorf 5804) and the pellet was discarded. The soluble fraction was loaded on the top of the sucrose gradient (20-60%; in buffer containing 20mM Tris, 2mM EDTA, 5% glycerol, 0.5% TX-100, pH 8.0) and centrifugated in Beckman SW32 rotor for 6h at 25500 rpm at 18°C. The gradient fractions (6 ml) were then removed from the bottom of the 38 ml tube. The CuMV VLP containing fraction (40 and 50% sucrose, pooled) was diluted 1:1 with buffer (20mM Tris, 2mM EDTA, 5% glycerol, pH 8.0). The VLPs were sedimented using Type 70 rotor (Beckman, 50000 rpm, 4h, 4°C). Then the pellet was dissolved ON in 4 ml of 20mM Tris, 2mM EDTA at 4°C. The solution was clarified by centrifugation (5min, 14000 rpm), the clarified solution overlaid on top of the 30% sucrose „cushion“ solution in 20mM Tris, 2mM EDTA, 0.5% TX-100, pH 8.0 The VLPs were sedimented using Beckman TLA100.3 rotor (72000 rpm, 60min, 4°C). The pellet was solubilized in 2 ml of 20mM Tris, 2mM EDTA and clarified again by centrifugation (5min, 14000 rpm). Obtained VLPs were characterized using SDS-PAGE, agarose gel, electron microscopy and dynamic light scattering. Protein concentration was determined using BCA test.