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).