2.4 Preparation of anti-NTD of S protein polyclonal antibody
All animal procedures were approved by the Ethics Committee of Henan Agricultural University. Two groups of eight-week-old BALB/c mice were respectively immunized subcutaneously three times, each at 2-week intervals, with 100 μg/mouse of recombinant G1-NTD and G2-NTD formulated in Freund’s complete adjuvant for the first time, 100 μg/mouse of corresponding recombinant protein formulated in Freund’s incomplete adjuvant for the second time, and 100 μg/mouse of corresponding recombinant protein for the third time. Two weeks after the third immunization, blood was collected from tail vein of mice from each group and pooled, and serum samples were prepared and store at -20 ºC until use.
2.5 Western blot
Same amount of purified G1-NTD and G2-NTD (200 ng/each) were subjected to 12% SDS-PAGE and transferred to a nitrocellulose (NC) membrane for 45 min. The NC membrane was blocked overnight at 4 ºC using 5% non-fat dry milk in PBS-0.05% Tween-20 (PBST) followed by incubation with the G1- or G2-NTD antisera (1:2000 dilution in PBST) at 37 ºC for 1 h with gentle rotating. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 dilution in PBST) at 37 ºC for 1 h, after complete washing with PBST. The protein bands were visualized using Novex® ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific).