Protein expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and purification of the three recombinant proteins was achieved through nickel affinity chromatography with the HisTrapTM
HP column. Each purified protein migrated as a single band with the predicted size in SDS-PAGE, of which purity was more than 95% (Figure 1). The specifiCity of the bands was confirmed by using specific antibodies generated against native forms of Prn, Fim2 or Fim3, respectively, in Western blotting (Figure 1). By using this approach, a large amount of proteins was obtained, at approximately 12 mg/L of rPrn, 25 mg/L of rFim2, and 19 mg/L of rFim3. Figure 1 SDS-PAGE and Western blotting analysis. (A) SDS-PAGE of the purified recombinant proteins. The proteins were electrophoresed on a 10% SDS-PAGE gel under reducing condition and Selleck Inhibitor Library stained by Coomassie blue. Lane 1: Molecular mass marker, the molecular mass Belnacasan in vitro standards are
indicated in kDa on left Selumetinib in vitro side; lane 2: rPrn (10 μg); lane3: rFim2 (10 μg); lane 4: rFim3 (10 μg). (B) Western blotting of the recombinant proteins. Lane 1: Pre-stained molecular mass marker (170 kDa, 130, 100, 70, 55, 40, 35, 25, 15, 10, Fermentas), the molecular mass standards are indicated in kDa on left side; lane 2: rFim2 was detected with mouse anti-Fim2 monoclonal antibodies; lane 3: rFim3 was detected with mouse anti-Fim3 monoclonal antibodies; lane 4: Pre-stained molecular mass marker, the molecular mass standards are indicated in kDa on right side; lane 5: rPrn was detected with mouse anti-Prn monoclonal antibodies; lane 6: Pre-stained molecular mass marker, the molecular mass standards are indicated in kDa on right side. Serum antibody responses
to rPrn, rFim2 and rFim3 In order to examine the antibody responses to rPrn, rFim2 and rFim3, sera of immunized mice were collected two weeks after the second immunization. Titres of serum IgG antibodies were measured by ELISA. Significant IgG antibody responses were observed in the mice immunized Rucaparib with both high and low doses of rPrn, rFim2 or rFim3 when compared to the control group (P < 0.001 for all three proteins) (Figure 2). High levels of IgG antibodies were induced in mice immunized with high doses of the three proteins. However, the differences were not significant when compared to those in mice immunized with low doses (Figure 2). When the same amount of rFim2 and rFim3 was used in immunization, IgG responses appeared to be similar between the two groups (P = 0.056). Figure 2 Antibody responses in immunized and control mice. Two weeks after the second immunization, sera were collected, and IgG antibody titres were determined by ELISA. Results represent the mean antibody titres for five mice per group. An asterisk symbol (*) indicates a statistically significant difference (P < 0.001) between immunized and control group.