The suspension was centrifuged, and the sediment was washed and then lysed in TE buffer containing urea. Proteins selleck inhibitor were purified on a 10-mL Source™ 30 Q anion exchange chromatography column (GE Healthcare Bio-Sciences, Uppsala, Sweden) using ÄKTA™
purifier systems (GE Healthcare Limited, Buckinghamshire, UK). The flow-through fraction containing Ag85b was collected, and the protein was refolded by gradient dialysis in TE buffer. For HspX, cells were lysed in TE buffer and sonicated. After centrifugation, supernatants were collected and purified on a 20-mL Q Sepharose high performance anion exchange chromatography column (GE Healthcare Bio-Sciences), and then the column was eluted stepwise with 15%, 50% and 100% v/v selleck compound TE buffer/1 M NaCl. The elution at 50% was collected and further purified by 40% ammonium sulfate precipitation. The supernatant was purified in the second step on a 20-mL phenyl-sepharose high performance
column (GE Healthcare Bio-Sciences) and eluted separately with 60%, 80% and 100% of TE buffer, and the eluate at 100% was collected and dialyzed to phosphate-buffered saline (PBS) buffer. The purified proteins were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and protein sequencing (15 amino acids of N-terminals) (National Laboratory of Medical Molecular Biology of Chinese Academy of Medical Sciences, Beijing, China). Both CpG DNA (1.78 mg mL−1) and aluminum hydroxide (11.98 mg mL−1) used in this study were obtained from Mycobacterium Laboratory of NICPBP. Vaccines were prepared by mixing Ag85b, HspX and recombinant C/E and combining it with either CpG or aluminum hydroxide or the mixture of CpG and aluminum hydroxide. All the guinea pigs were divided into five groups (with 12 animals in each group) according to vaccine combinations as follows: group Ag (Ag85b, HspX and C/E; 10 μg of each protein per animal), group Ag+Al
(Ag85b, HspX, C/E and aluminum, 10 μg of each protein and 0.35 mg of aluminum per animal), group Ag+Al+CpG (Ag85b, HspX, C/E, aluminum and CpG; 10 μg of each protein, 0.35 mg of aluminum and 75 μg of CpG per animal), group Ag+CpG (including Ag85b, HspX, Casein kinase 1 C/E and CpG; 10 μg of each protein and 75 μg of CpG per animal) and group nonstimulated (NS) (0.2 mL of natural saline per animal). Each guinea pig was challenged by subcutaneous injection of Mtb H37Rv at a dose of 1150 CFU on the inner side of a hind leg. Five days after challenge, the animals were vaccinated with freshly prepared vaccines injected by the intramuscular route three times at an interval of 2 weeks, and the negative control group was vaccinated with natural saline. Animals were sacrificed 2 weeks after the last vaccination and then assayed for lung, liver and spleen lesion scores and spleen bacterial loads.