Expression and purification of 30 kilodalton protein antigen of Ara- Burkholderia pseudomallei.

The 30 kDa protein of B. pseudomallei is found in virulent Ara- but not avirulent Ara+ strain. The gene was cloned in Escherichia coli JM105 employing pInIII-C2 vector. The open reading frame was 870 nucleotides with a guanine plus cytosine content of 69.9%. Arginine was the most abundant amino acid in the protein, having a PI of 12.65. Nucleotide sequence of the gene was 96% identical to B. pseudomallei 1710b chromosome II sequence CP000125.1, encoding an oxidoreductase of the short chain dehydrogenase/reductase family. The 30 kDa antigen was expressed as a maltose-fusion protein with a yield of 5.25 mg/l of bacterial culture.

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.