To prepare crude extract of C. parvum, 2·3 × 107 purified oocysts were resuspended in 1·5 mL PBS (0·05 m, pH 7·4), frozen in liquid nitrogen for 5 min and melted at 23°C for 10 min for three times. The freeze-thawed oocyst suspension was sonicated at 300 W for 40 min, centrifuged

at 3000 × g 10 min and the supernatant was collected Y-27632 cost and stored at −80°C until application in the subsequent experiments. To prepare the recombinant proteins, the above plasmids were transformed into Escherichia coli BL21 (DE3) and the expression of proteins was induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) at final concentration of 1 mm for 5 h. The cells were collected by centrifugation at 10 000 × g, 4°C for 10 min and the pellets were resuspended in NTA-0 Buffer (20 mm Tris–HCl, pH 7·9, 0·5 m NaCl, 10% glycerol, and PMSF, lysozyme 0·2–0·4 mg/mL). After incubation on ice for 30 min, the cells were sonicated for 10 min, followed

by the incubation with 0·05% Triton X-100 on ice for 15 min, 1 mm MgCl2, DNase I 10 μg/mL at room temperature (RT) for 10 min. After centrifugation at 10 000 × g, 4°C for 15 min, the supernatant was collected. To obtain right refolding protein, the recombinant protein was dialysed in PBS (0·05 m, pH 7·2) for 3 days, then in the solution of 0·5 m urea, 20 mm Tris–HCl, pH 8·0, 1 mm EDTA for 24 h, in the solution of 20 mm Tris–HCl, pH 8·3, 1 mm EDTA, 2 mm reduced glutathione, 0·2 mm oxyclozanide l-glutathione oxidized for 24 h. After concentration with PEG8000, the protein was resuspended in PBS for GW-572016 supplier the subsequent experiments. Inbred BALB/c healthy mice, age 4–6 week-old, without other intestinal parasite infection (excluded via stool examination with Ziehl-Neelsen stain) were selected and randomly divided into different groups. The selected mice were immunized subcutaneously with 10 μg proteins diluted with sterilized normal saline and emulsified in complete Freund’s adjuvant (Gibco BRL, Grand

Island, NY, USA). Subsequent immunizations on days 14 and 28 post-immunization were performed with the same dose of protein in incomplete Freund’s adjuvant. A control group of mice were given adjuvant alone. Blood samples of mice were collected from the retro-orbital plexus at baseline 2 weeks after each immunization. Serum immunoglobulin G (IgG) antibody response specific to differently prepared C. parvum antigens were measured by ELISA as previously described (14). Briefly, flat-bottom 96-well ELISA plates were coated with 0·15 μg/mL of antigen in 0·1 m carbonate buffer (pH 9·6) 50 μL per well and incubated overnight at 4°C. The plates were blocked with 3% bovine serum albumin (BSA)–PBS containing 0·3% Tween-20 for 1 h at 4°C. After washing, 50 μL of serial diluted serum sample in 0·05% Tween 20-PBS was applied to the wells in duplicate and the plates were incubated for 2 h at RT.