The protein was purified under native conditions. Briefly, a column was equilibrated with native buffer (20 mM sodium phosphate containing 0.5 M NaCl,
pH 7.4). Then, the protein click here preparation was applied to the column and eluted gradiently from 20 to 500 mM imidazole (pH 7.4). SDS-PAGE described below was used to identify the fractions containing the desired protein and to determine their purity. Finally, elution product by 500 mM imidazole was used for the experiment and the protein concentration was estimated using a BCA protein assay kit (Bio-Rad, Rockford, IL, USA). Sodium dodecylsulfate PAGE analysis was performed with the DYY-5 protein electrophoresis system (12 cm × 8 cm × 0.75 cm; Beijing Kesheng, Beijing, China). The stacking and separation gels contained 5%
and 10% acrylamide, respectively. Electrophoresis was carried out at a constant voltage of 100 V for almost 180 min. Then, the gels were either stained with Coomassie blue R or electroblotted onto nitrocellulose membranes. MALDI-TOF-MS (Applied Biosystems, Foster City, CA, USA) was performed after SDS-PAGE by the Department of Diagnosis (National Institute for Communicable Disease Control and Prevention, China CDC). Briefly, the expressed protein was excised from the gels, and in-gel digestion by trypsin performed. The resulting tryptic peptides were analyzed with an ABI 4700 MALDI-TOF-MS, AUY-922 following searching against the National Center for Biotechnology Information database. Specificity of the recombinant protein was assessed by ELISA with rabbit sera against common members of the order Rickettsiae (C. burnetii; R. mooseri; R. heilongjiangensis; R. prowazekii; R. conorii; O. tsutsugamushi strain TA763; O. tsutsugamushi strain Karp; O. tsutsugamushi
strain Kato; R. parkeri; O. tsutsugamushi strain TH1817; B. bacilliformis; E. chaffeensis; B. quintana; R. australis; A. phagocytophilum; R. sibirica) which were previously prepared in our laboratory. Specificity was also tested with rabbit sera against genetically unrelated pathogenic bacteria, including L. pneumophila; Haemophilus; N. meningitidis group C; N. meningitidis group Y; N. meningitidis group B; N. meningitidis Sucrase group A; N. meningitidis group W135; S. dysenteriae and Y. enterocolitica, which were supplied by related laboratories of the National Institute for Communicable Disease Control and Prevention, China CDC. Enzyme-linked immunoassay was performed as described previously (15). Briefly, polystyrene 96-well microtiter plates (Corning Glass, Corning, NY, USA) were coated overnight at 4°C by adding 100 μL antigens (recombinant 56-kDa protein diluted in PBS; final concentration, 0.5 ng/mL) and blocked with 10% BSA for 1 hr, and rinsed four times with PBS for 3 min each time. Twofold serially diluted rabbit antisera were added to the ELISA plates.