Ampicillin(100 μg/mL) was used for plasmid selection. Dot blotting was used to detect proteins that induce antibody responses during S. aureus USA300 Pifithrin-�� supplier infection. 19 recombinant proteins associated with S. aureus virulence were purified in our lab before and were dotted onto nitrocellular membrane before the membrane was air dried. The membrane was then washed three times with PBS containing 0.05% Tween-20 and blocked with PBS containing 5% milk at room temperature for 1 h. Sera from BALB/c mice infected with S. aureus USA300, 546 or 1884 were diluted with the blocking solution and incubated with

the membrane at room temperature for 1 h. After washed 3 times with PBST, the membrane was then incubated with HRP-labeled goat-anti-mouse IgG at room temperature for 1 h. After washed with PBST for 3 times, the membrane was developed with ECL substrate. A fragment of sasA gene, named fSasA, was amplified by PCR from S. aureus USA300 using the following PCR primers and standard PCR amplification conditions: sasAF(5′-CGCATATGAGTCATAGTTTAGTGAGTCAAGA-3′) and sasAR(5′-TTCTCGAGGATACCATCTCCACCATTT-3′).

The PCR product was cloned into pET21a(+) using the protocol described by the manufacturer (Novagen) and transformed into E. coli (DE3) cells. Two liters of fSasA-producing cells were grown in a 5-liter flask with constant shaking until A600 reached 0.8. Cells were harvested 4h after IPTG (1mM) induction by centrifugation (9,000 × see more g, 4 °C, 20 min). Cell pellets were suspended in a solution containing 25mM Tris-HCl (pH 8.0) and lysed by sonication. The lysate was clarified by centrifugation (12,000 × g, 4 °C, 30 min). His-tagged fSasA was purified from the clarified lysate first by Q Sepharose Fast Flow (GE) chromatography and then by HisTrap HP (GE) chromatography. The column was washed with

buffer A which contains 20 mM sodium phosphate, 0.5 M NaCl, 25 mM imidazole (pH 7.4), and His-tagged fSasA was eluted with a linear gradient from buffer A to buffer B which contains 500 mM imidazole, 20 selleck products mM sodium phosphate, 0.5 M NaCl (pH 7.4). The HisTrap HP eluate was concentrated and exchanged to PBS buffer by ultrafiltration using Amicon ULTRA-15 centrifugal filter devices (Millipore). BALB/c mice (20– 25 g, inbred, females) were immunized introperitoneally with 20 μg of purified fSasA protein absorbed on aluminium hydroxide and boosted two times at day 14 and day 28 after first immunization. Blood samples were drawn 7 days after the second and third immunizations and specific serum IgG levels were determined by ELISA. Mice were challenged with 3 × 109 S. aureus USA300 or 5 × 108 S. aureus 546 by intraperitoneal injection 35 days after the primary immunization and were monitored for 7 days. The specific binding of purified fSasA to serum from BALB/c mice immunized with fSasA was tested by ELISA. Briefly, fSasA was coated onto 96-well microtiter plates (0.3 μg/well) overnight at 4 °C.