Then sera from immunized mice were diluted before added into the wells and incubated for 2 h at 37 °C. Plates were then washed with washing buffer (PBS-0.05% Tween 20) three times for 3 min each and goat anti-mouse IgG was added into the wells and incubated for 1 h at 37 °C. After washing as above, TMB (3, 3′,5,5′-tetramethylbenzidine dihydrochloride) substrate
(Sigma) was added and color intensity was determined spectrophotometrically at OD 450 nm. Statistical analysis was performed by Gehan-Breslow-Wilcoxon Test using Graphpad Prism 5. P≤ 0.05 was regarded as significant. 19 proteins associated with S. aureus invasion or pathogenesis were dotted onto NC membranes and reacted with sera from mice recovered from infection with different S. aureus strains. The sera from learn more mice infected with S. aureus 1884 reacted with proteins FnBA, SasA, SasF and SPA (S. aureus proteins A) (Fig. 1A). The sera from mice infected with S. aureus 546 reacted with proteins
CNA, FnBA, SasA, SasF, and SPA(Fig. 1B). The sera from mice infected with S. aureus USA300 reacted with proteins ClfA, IsdA, SasA and SPA (Fig. 1C). We found different S. aureus strains induced different antibody responses. The proteins SasA and SPA reacted with all of the sera. Protein SPA is a mitogen that interacts with many immunoglobulins by binding to the Fc region. The results showed that SasA was expressed on all of the above strains and could induce strong antibody response during S. aureus infection. Ku-0059436 concentration To detect whether the antibody response induced by SasA is protective, part of the protein was expressed. The SasA protein is composed of 2272 amino acids. The secondary structure of SasA protein was analyzed by DNAstar software and fragment (48aa-333aa, named fSasA) was selected to be amplified from the genomic DNA of S. aureus USA300 by PCR using primers SasAF and SasAR. Recombinant plasmid pET-fSasA was constructed, sequencing verified, and transformed into E. coli BL21 for protein expression. After induction with 1 mM IPTG at 37 °C for 4 h, the total soluble proteins of bacteria were analyzed by SDS-PAGE. It showed
that fSasA was expressed at a level of up to 10% of whole cell protein (Fig. 2A). After 2-step purification, fSasA protein of high purity was obtained (Fig. 2B). Western blot with antibody against 6x His tag showed that Vorinostat in vitro the protein size was correct (Fig 2C). The purified protein can be used as antigen for animal immunization. SasA is a surface protein of S. aureus. The fSasA was absorbed well by aluminium hydroxide gel in physiological saline. After second immunization, BALB/c mice generated strong IgG response against fSasA protein. The response was further elevated after third immunization (Fig. 3). To test the role of immunity induced by fSasA against S. aureus infection, BALB/c mice were challenged with 3 × 109 S. aureus USA300, collected at early exponential phase, 7 days after the third immunization with fSasA.