The E. coli pellet was suspended and sonicated. The supernatant and precipitate were separated

by centrifugation. To purify r3aB, the supernatant was directly loaded onto a Ni-NTA column (Pharmacia Biotech) to remove almost all of the bacterial proteins. To purify r3AB, the precipitate of cell lysate was collected and dissolved by 8 mol/l urea and then centrifuged. Cyclopamine chemical structure The resultant supernatant was loaded onto Ni-NTA column to remove bacterial proteins. The bound r3AB or r3aB was eluted and then loaded onto Sephadex G-25 column to remove imidazole and change the buffer to saline. The products were analyzed on 12% SDS-PAGE. The r3aB and r3AB at 8 μg/ml were coated on 96-well polystyrene microtiter plates (Yangzi Company, Jiangshu, China), and incubated overnight at 4 °C in 0.01 mol/l PBS (1 mmol/l KH2PO4, 10 mmol/l Na2HPO4, 137 mmol/l NaCl, 2.7 mmol/l KCl, pH 7.4). After washing for three times with washing buffer (PBST, 0.01 mol/l PBS,

0.05% Tween 20), 200 μl of blocking buffer was added (0.01 mol/l PBS, 5% skim milk) followed by incubating at 37 °C for 2 h. The sera were diluted at 1:100 with sample buffer (0.01 M PBST, 5% skim milk), added to wells in duplicate and incubated at 37 °C for 1 h. Afterwards, plates were washed three times followed by the addition of 100 μl Onalespib nmr per well of rabbit anti-bovine IgG/HRP (Sigma) at 1:5000 dilution and incubation at 37 °C for 1 h. After washing three times, the substrate solution of Ophenylenediamine dihydrochloride (OPD) (Amresco) was added and incubated at room temperature for 5 min for color development which was stopped with 50 μl per well of 2 M sulphuric acid. The optical density (OD) of the color in each well of plates was determined at 492 nm on an automated ELISA plate reader. The results were expressed as A492 ± SD. To obtain coating antigens for establishing indirect ELISAs to detect FMDV NSP-specific antibodies ADP ribosylation factor in cattle, recombinant 3AB (r3AB) was expressed in E. coli. The cells expressed r3AB were collected and subsequently sonicated. After separation by

centrifugation, the supernatant and precipitate were analyzed by SDS-PAGE. As shown in Fig. 1a, an abundant band with 37 kDa was revealed in the lane loaded with the precipitate, indicating that r3AB was majorly expressed in inclusion body. To purify the r3AB, the inclusion body was broken by lysis buffer containing 8 mol/l urea and the expressed proteins experienced refolding process by reducing the amount of urea. After purification, the r3AB displayed two bands close to 74 kDa and 37 kDa by SDS-PAGE, indicating that the purified r3AB existed as a mixture of monomers and dimers ( Fig. 1b). To avoid the inclusion body formation and dimers aggregation, a gene coding a truncated 3AB protein (r3aB) by deleting 80 amino acids at N-terminal of 3AB was constructed (Fig. 2a). The only cysteine at 65th residue was eliminated by the deletion.