tuberculosis H37Rv chromosomal DNA template with the primers Ag85BF, 5′-AATGGATCCTTCTCCCGGCCGGGGCT-3′(BamHI), and HspXF1, 5′- ATAGAGCTCATGGCCACACCCTTC-3′(SacI), as forward primers and the primers Ag85BR, 5′-ATTGAGCTCGCCGGCGCCTAACGAACTCTGGAG-3′(SacI), and HspXR, 5′-ACGAAGCTTTCAGTTGGTGGACCG-3′(HindIII), as reverse primers. The PCR fragment of Ag85B was cloned into the BamHI and SacI site of pET-28a to construct the plasmid pET-28a Ag85B. Subsequently, the fragment of Mpt64190–198-HspX
was generated by PCR from PCR product of HspX as template with the forward primer HspXF2, 5′-ATAGAGCTCTTCGCAGTCACGAACGACGGGGTGATTATGGCCACCACCCTTC-3′(SacI), and the reverse primer HspXR and was cloned into the unique site SacI and HindIII of the previously constructed pET-28a Ag85B plasmid. The correct DNA construct containing the appropriate inserts was confirmed PD98059 by DNA sequencing. Expression and purification of AMH fusion protein. The plasmid pET-28a AMH was transformed into the Escherichia coli strain BL21 for Compound Library production of the fusion protein AMH. E. coli BL21 expressing AMH was cultured in LB medium for 2 h at 37 °C
before induction with 1 mm isopropylβ-d-thiogalactopyranoside. After induction, growth was continued for 4 h at 37 °C when the bacterial cells were harvested by centrifugation at 12,000 g for 10 min at 4 °C. Then, cells were suspended in buffer A without urea (sodium phosphate buffer 0.1 m, Tris–Cl 0.01 m, pH 8.0) at 5 ml per gram wet weight and sonicated on ice at 200–300 W for 30 min Adenosine triphosphate with 1-s cooling period between each 1-s bursting. The insoluble material containing AMH in inclusion bodies was precipitated by centrifugation at 12,000 g for 10 min at 4 °C, and AMH was solubilized and extracted overnight at 4 °C in buffer B (urea 8 m, sodium phosphate buffer 0.1 m, Tris–Cl 0.01 m, pH 8.0). AMH protein was subsequently purified by Ni-NTA His resin-columns (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s instructions. Fractions containing AMH were identified by 12% SDS-PAGE and pooled. Finally, the pooled fractions were dialysed against urea
concentration gradient (6, 4, 2, 1, 0.5 and 0 m urea with 5 mm Tris–Cl, pH 7.9) for 12 h at each concentration at 4 °C. The concentration of the pure AMH was determined by the Lowry protein assay. Subunit vaccine preparation. AMM, Ag85B and BCG PSN were prepared as described previously . The preparation of AMH and AMM + AMH vaccines is described below. AMM and AMH were suspended in phosphate-buffered saline (PBS) (0.2 mg/ml), and BCG PSN was suspended in saline (0.6 mg/ml). DDA (Sigma-Aldrich, St. Louis, MO, USA) was suspended in distilled water (2.5 mg/ml), and a homogeneous dispersion of the powder was obtained by heating the suspension to 80 °C for 5–10 min. After cooling at room temperature, the suspension was mixed with diluted AMM, AMH and BCG PSN before use. Vaccination and challenge procedures.