Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen

Tucci, Paula; Estevez, Verónica; Becco, Lorena; Cabrera-Cabrera, Florencia; Grotiuz, Germán; Reolon, Eduardo; Marín, Mónica

Culture was centrifuged at 10000×g and the cell pellet was stored at −70°C. M. haemolytica genomic DNA was extracted using a standard protocol (Ausubel et al., 1999) and partial LKT sequence was amplified by PCR. For LKT expression, restriction sites were included in primers for a directional cloning of LKT sequence in pET28a(+) vector (Novagen). E. coli BL21 DE3 (Novagen) competent cells were transformed with the construct and stored at −70°C in 15% glycerol. Plasmid DNA was isolated from E. coli using a commercial kit (QiaPrep Spin Miniprep, Qiagen). Plasmid sequence was confirmed by DNA sequencing at Institut Pasteur Montevideo (Uruguay). For LKT expression, a culture was performed in ZYM-5052 auto induction medium (Studier, 2005) for 18-20 h at 37.0 ± 1.0°C. As expected, by SDS-PAGE a band of approximately 40 kDa corresponding to the recombinant protein was observed. The protein was purified by IMAC chromatography (HisTrap HP, GE Healthcare) in an ÄKTA Purifier 100 (GE Healthcare). The Article No~e00158 purified protein was dialyzed against PBS 1X pH 7.4 and quantified by BCA (Pierce BCA Protein Assay Kit, Thermo Scientific).