Table 1.
Primer list.
Table 2.
In silico selected CDSs for identification of immunoreactivity.
Fig 1.
Expression and immunoreactivity of candidate proteins.
(A) Coomassie brilliant blue (CBB) staining and (B–D) Western blotting of the proteins in cell pellet fractions of the control and candidate protein-expressing Escherichia coli strains grown in terrific broth. In Western blotting, the proteins in the cell pellets were probed with different antibodies: (B) anti-glutathione S-transferase (GST) immunoglobulin G; (C) horse hyperimmunized anti-Bacillus anthracis serum (PC1); (D) naive horse serum (NC1). Lane 1, Mw, molecular weight marker (in kDa); lane 2, control strain which is E. coli BL21 harboring empty pGEX-6P-2 without IPTG induction; lane 3, control strain which is E. coli BL21 harboring empty pGEX-6P-2 with 0.2 mM IPTG induction; lane 4, E. coli BTZ001 expressing recombinant hypothetical protein (GST-GBAA_RS28110: 44 kDa); lane 5, E. coli BTZ002 expressing recombinant capsule biosynthesis protein CapA (GST-GBAA_RS28240: 72 kDa); lane 6, E. coli BTZ003 expressing recombinant signal peptidase (GST-GBAA_RS28275: 47 kDa); lane 7, E. coli BTZ004 expressing recombinant peptide ABC substrate-binding protein (GST-GBAA_RS28340: 84 kDa); lane 8, E. coli BTZ005 expressing recombinant metal-dependent hydrolase (GST-GBAA_RS28430: 46 kDa). The arrows indicate target proteins in the expected sizes.
Table 3.
Constructs and strain list.
Table 4.
Sequence similarity of two immunoreactive proteins identified by Western blotting with horse hyperimmune antisera and CapA322.
Fig 2.
Expression and purification of CapA322.
(A) Genetic map of Bacillus anthracis capsule-coding operon revealing the CapA322, C-terminus region (amino-acid 322–411) of CapA used in antigen preparation. (B) Coomassie brilliant blue staining (CBB) and (C, D, and G) Western blotting of the fractions collected after affinity batch purification. In Western blotting, proteins were probed with different horse sera: (C) horse hyperimmunized anti-B. anthracis serum (PC1); (D) naive horse serum (NC1); (G) vaccinated horse serum (Vac_H3D21). Lane 1, Mw, molecular weight marker (in kDa); lane 2, the supernatant fraction applied to affinity beads (GST-CapA322: 37 kDa); lane 3, beads bound; lane 4, elute after treatment with PreScission Protease (CapA322: 11 kDa). (E and F) CBB and Western blotting of the fractions collected during the cation exchange process. Lane 1, Mw, molecular weight marker (in kDa); lane 2, sample loaded onto the cation exchange chromatography column; lanes 3 and 4 flow-throughs; lane 5, the eluted protein. Host cell-derived contaminants indicated as Escherichia coli’s protein.
Fig 3.
Horses anti-PAD1 immunoglobulin G (IgG) responses against subcutaneously injected Bacillus anthracis Sterne 34F2 strain spore vaccine.
(A) anti-PAD1 IgG response of vaccinated horses in serum dilution 1:100. Each serum sample was tested in technical triplicate by PAD1-ELISA. Checkerboard titration between PAD1 and (B) horse hyperimmunized anti-B. anthracis serum (PC1) or (C) naive horse serum (NC1) in PAD1-ELISA. Each dilution of serum sample was tested in technical triplicate. The twofold serum dilution starts with a dilution of 1:100, and PAD1 dilution starts with 1.6 μg/well. From the result, we determined the optimal concentrations of the antigen, antibody, and serum dilutions as follows: antigen, 0.4 μg/well; serum dilution, 1:100; second antibody dilution, 1:15,000.
Fig 4.
Comparison of PAD1-ELISA and CapA322-ELISA.
Checkerboard titration between CapA322 and (A) horse hyperimmunized anti-Bacillus anthracis serum (PC1) or (B) naive horse serum (NC1) in CapA322-ELISA. Each dilution of serum sample was tested in technical triplicate by CapA322-ELISA. The twofold serum dilution starts with a dilution of 1:100, and CapA322 dilution starts with 1.6 μg/well. From the result, we determined the optimal concentrations of antigen, antibody, and serum dilutions as follows: antigen, 0.8 μg/well; serum dilution, 1:100; second antibody dilution, 1:15,000. (C) Comparison of PAD1-ELISA and CapA322-ELISA on horse sera. Each serum samples of horse hyperimmunized anti-Bacillus anthracis (PC1), horse naturally infected with B. anthracis (PC2 and PC3), naive horses (NC1 and NC2), and horse vaccinated with Sterne34F2 strain (Vac_H1D21-Vac_H4D21) was analyzed in technical triplicate by PAD1-ELISA and CapA322-ELISA. (D) One-way ANOVA with Tukey’s multiple comparison test on relative OD values of positive (PC; n = 3), negative (NC; n = 2), and vaccinated (Vac; n = 3) groups.