The calculated M r s were: ABC transporter Abc, M r ~33 kDa; GapN, M r ~26 kDa; GlpO, M r ~41 kDa; and LppB, M r 43 ~kDa (compare with Figure 3). PtsG was isolated from the soluble fraction using nickel chelation, but it manifested in PAGE as two bands with M r s ~70 and ~45 kDa (Figure 3; calculated M r ~28 kDa). Figure 3 Expression of the five

selected proteins in E. coli. SDS-PAGE (10%) showing segments BIIB057 datasheet of the protein antigens that were expressed in E. coli. Lanes: M, molecular mass standards; 1, 12 μg of total antigen of MmmSC strain 8740; 2-6, expressed segments of proteins Abc, GapN, GlpO, LppB and PtsG, respectively. The pool of the seven sera obtained from the Botswana outbreak was also used in immunoblotting. The pool reacted with the

expressed Abc and LppB polypeptides (Figure 4). The PtsG polypeptide bands were probed separately with serum obtained from an experimental infection. This immunoblot, however, showed multiple bands that apparently reacted with the pooled sera (not shown). Figure 4 Chemiluminescent immunoblot. Recognition of the ABC transporter Abc (lane 1) and Selleck KU57788 lipoprotein LppB (lane 4) polypeptides that were expressed in E. coli by a pool of sera obtained from cattle that were naturally infected AZD9291 supplier with CBPP during the 1995 Botswana outbreak. The GapN (lane 2) and GlpO (lane 3) polypeptides were not recognised in this test format. Discussion When a pathogen infects an animal, its epitopes leave CYTH4 an “”imprint”" in the form of a spectrum of disease-specific antibody paratopes

in the serum. Most animals are therefore likely to have antibodies directed against a large number of foreign epitopes. The strategy pursued in this study was to use this complex mixture of antibodies to select binders from a limited repertoire of sequences derived from the genome of MmmSC, thereby focussing the phage display selection process on relevant epitopes. These binders were matched to open reading frames present in the genome. Unlike immunoblotting, this approach also identified the genes that coded for the antigenic proteins. The fragmented genome library covered approximately 97% of the mycoplasmal genome. While adequate for its purpose, it cannot, however, be considered to have been completely random since among the 1016 proteins encoded in the genome of MmmSC type strain PG1, 797 (78.4%) contain at least one UGAtrp codon, which is read as stop codon in E. coli. Moreover, the frequency of UGAtrp codons in coding sequences of MmmSC genes is relatively high: 1.00% in contrast to 0.05% of UGGtrp codons. This means that epitopes containing such stops could be disrupted. Moreover, in a phage display system, the secreted phages would be unlikely to display large oligopeptides or those that resisted being transported through the bacterial membrane or periplasm.