Expression and purification of the recombinant S protein
For recombinant proteins expression, HEK 293F cells were cultured at an orbital shaker incubator in two 1 L flasks containing 500 mL culture volume until the cell density reached 2 × 106cells/mL, and the cell survival rate was above 95%, then infected with recombinant or wild-type baculoviruses (rB or wB) at a multiplicity of infection (MOI) of 20 and incubated at 37 ℃. After 8 h, sodium butyrate (Shanghai Macklin Biochemical Co., Ltd, China) was added to the final concentration of 10 mM, and incubated for 3 days at 37 ℃ in 5% CO2. Cells were then collected, lysed in 1% Triton X-100, and then centrifuged at 12000 × g for 30 min at 4 ℃. The supernatants were prepared and examined with Western Blot as described below.
The resulting precipitates were resuspended in 1 × phosphate buffer saline (PBS) (pH = 7.4), ultrasonicated, and then centrifuged at 12000 ×g for 10 min at 4 ℃. The supernatants were collected and purified using protein G REsin (GenScript, Nanjing, China) according to the manufacturer’s instructions. The purified proteins were analyzed using Coomassie Blue stained SDS-PAGE electrophoretogram, then quantified by NanoDrop spectrophotometry (IMPLEN, Germany) and supplemented with 1% protease inhibitor cocktail C (Beijing Yataihengxin Company, China) and frozen at −80 °C until use.