All panels were characterized by clinical examination, parasitology, serology and PCR. In addition, the sera were characterized as positive for other agents by clinical examination and serological tests. Samples of other canine diseases were as follows: 14 for Trypanosoma caninum, 34 for Leishmania brasiliensis, 20 for Babesia canis and 18 for Ehrlichia canis. All
sera were collected in the fieldwork and were characterized in reference centres of the regions mentioned above. The proteins rLci2B and rLci1A were cloned in pRSET B and pBK-CMV, respectively. All constructs were obtained from the Laboratory of Pathology and Biointervention (Laboratório de Patologia e Biointervenção, CPqGM, FIOCRUZ/BA, Brazil). The E. coli, strain BL21 (DE3)/pLysS, was transformed with those plasmids. Fermentation was carried out in Luria Broth medium
with ampicillin (100 μg/mL) at 37°C NVP-BGJ398 until the absorbance at 600 nm reached 0·6. Recombinant protein expression was induced by the addition of 1 mm isopropyl-β-d-thiogalactopyranosid. During fermentation, samples were collected Ku-0059436 supplier at regular time intervals to check the protein expression by SDS-PAGE. Four hours after induction, cells were harvested by centrifugation, collected and lysed by sonication in 20 mm sodium phosphate buffer with 150 mm NaCl, pH 8·0, containing 5 mm lysozyme, and 1 mm phenylmethane-sulphonylfluoride. The protein rLci2B was recovered from the soluble fraction (crude extract I), while the rLci1A present in inclusion bodies required solubilization in 8 m urea (crude extract II) (24). After the expression and purification steps, the analysis of the recombinant proteins was carried out by polyacrylamide gel electrophoresis (T = 12%; C = 3%) under denaturing conditions according to Laemmli (25), in a vertical Mini Protean Idoxuridine III System (Bio-Rad Laboratories Inc. Hercules, CA, USA). The molecular weight protein markers (prestained broad range) were from
Bio-Rad Laboratories Inc. The protein bands were visualized after staining with 0·1% coomassie brilliant blue R-350 in a methanol/acetic acid/water (30 : 8 : 62, v/v/v) solution and destained by a methanol/acetic acid/water (30 : 10 : 60, v/v/v) solution. Crude extract I was submitted to immobilized metal affinity chromatography using a Ni-NTA Superflow agarose (Qiagen, Duesseldorf, Germany). The column was equilibrated with 20 mm sodium phosphate buffer, 150 mm NaCl, pH 8·0. The rLci2B was eluted with a step gradient containing 500 mm imidazol. The fractions containing rLci2B were pooled and applied onto a Superdex™ 200 (GE Healthcare, Little Chalfont, UK) previously equilibrated in 50 mm Tris–HCl, 150 mm NaCl, pH 8·0. Crude extract II was purified by anion ion-exchange chromatography (Poros® HQ; Applied Biosystems, Foster City, CA, USA) in the presence of 4 m urea.