Arabinose at concentrations from 0.02 to 0.2 μg mL−1 was added to LBA so as to induce the recombinant fusion protein. Various time periods of incubation at 37 °C under agitation were tested to determine the optimal expression conditions of the TbpA-His fusion protein. Thereafter, the cultures were centrifuged, bacteria were resuspended in lysis buffer and sonicated (three cycles of 20 s, 40% duty cycle, Branson sonifier 450 Branson, VWR, Spain) before being centrifuged. The protein concentration was measured from the supernatants obtained using Bradford’s method. These samples were then analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE). Immunoblots

were carried out as described previously www.selleckchem.com/products/bmn-673.html (Pyle & Schill, 1985) in order to confirm the TbpA-His fusion protein in these gels. The membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) for 2 h at 37 °C,

and incubated for 1 h at 37 °C with horseradish peroxidase-labeled murine anti-V5 monoclonal antibodies (mAbs) (Invitrogen) diluted 1 : 5000 in TBS. These mAbs recognize the V5 epitope, which is located in the learn more C-terminal domain of the protein fusion. Bound antibodies were detected adding an enhanced chemiluminescent substrate (GE Healthcare, Spain) (Bronstein et al., 1992). Nickel affinity chromatography (His-Select™ HC Nickel affinity gel, Invitrogen) was used for the purification of the TbpA-His fusion protein, which was eluted using a phosphate-buffered saline (PBS) buffer containing imidazole (from 75 to 250 mM). Crude extracts, unbound and eluted

fractions were analyzed by SDS-PAGE to monitor the optimal conditions for expression and purification. Five groups of two 3-month New Zealand rabbits (Charles River, Spain) were immunized with different rTbpA antigens: (a) minced pieces ASK1 of a Ponceau Red-stained nitrocellulose membrane containing a purified rTbpA fragment, (b) the same antigen as (a), but treating the nitrocellulose membrane with dimethyl sulfoxide, (c) small pieces of a minced Coomassie-blue-stained electrophoresis gel containing an rTbpA fragment band, (d) the purified protein extract, and (e) PBS. Fifty micrograms of each antigen was emulsified in Montanide IMS 2215 VG PR (Seppic Inc., France) at a 1 : 4 ratio and injected intramuscularly. Booster immunizations were administered 21, 42 and 63 days later in the same way, and rabbits were bled 7 days after the last injection. Sera were collected, inactivated at 56 °C for 30 min, adsorbed as reported earlier (del Río et al., 2005) for reducing background staining and stored at −80 °C until use. The animals were handled and cared in accordance with European Animal Care guidelines. Bacterial extracts containing iron-binding proteins from H. parasuis (Nagasaki), A. pleuropneumoniae (WF83) and S. aureus were obtained under iron-starved conditions using 2.2 dipyridyl (100 μM). These samples were analyzed by SDS-PAGE.