The PCR condition is as following: 30 cycles (94 °C 50 s, 66 °C 50 s, 72 °C 50 s),72 °C 10 min. Then, the cDNA of Ag85A was inserted into the BamHI and XbaI restriction sites of pcDNA3 plasmid (Invitrogen, Carlsbad, CA, USA), downstream of the CMV early promoter. For the construction of ubiquitin-Ag85A fusion DNA vaccine, the cDNA encoding the ubiquitin with HindIII and BamHI restriction sites was obtained from mouse testicle by RT-PCR. An arginine (R) was added to the C-terminal residues of Ub. The cDNA of Ag85A antigen with BamHI and XbaI restriction sites was also obtained by PCR, not including the starting codon. The spacer sequence (GGGGS) was

added between the ubiquitin and Ag85A antigen. Plasmids used in this study were prepared with alkaline Pexidartinib lysis method followed by TritonX-114 treatment to remove endotoxin [18]. Vaccination protocol.  For DNA vaccination, mice were injected with pcDNA3-Ag85A or pcDNA3-ub-Ag85A (UbGR-Ag85A) into both quadriceps with 2 × 50 μg DNA three times at 3-week intervals. Mice inoculated with pcDNA3 plasmid or pcDNA3-ub were used as negative controls. To enhance muscle cells uptake of plasmid DNA [19], 25% sucrose was injected into the muscles

of both quadriceps 15 min before plasmid inoculation. Enzyme-linked Immunoabsorbent assay (ELISA).  Anti-Ag85A IgG, IgG1 and IgG2a were measured by ELISA in individual serum sample from vaccinated mice. www.selleckchem.com/products/chir-99021-ct99021-hcl.html The method was as described previously [19], using recombinant Ag85A protein (1 μg per well) see more [20] and anti-mouse IgG, IgG1 or IgG2a coupled to horseradish peroxidase (HRP) (Southern Biotechnology Associates, SBA, Birmingham, AL, USA). The antibody titres were determined according

to the optical density (OD 450 nm). Finally, the relative ratio of IgG2a to IgG1 was calculated. Lymphocytes proliferation assay.  Mice were sacrificed 3 weeks after the last immunization. Spleens from each group were pooled and analysed. Th cell proliferation assay was performed as previously described [21]. Briefly, the isolated spleen cells were resuspended to a concentration of 5 × 106 cells/ml. A volume of 100 μl of cell suspension was added to 96-well plates, and the Ag85A protein [20] was added to the wells in triplicate at the final concentration of 5 μg/ml. The plates were incubated at 37 °C in an atmosphere of 5% CO2 for 66 h. Then the proliferation responses were detected by MTT [3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] (5 mg/ml; Sigma, St. Louis, MO, USA) method, and the stimulation index (SI) was calculated. The stimulation index was determined from the formula: stimulation index (SI) = experimental OD/negative OD. To assure that cells were healthy, 10 μg/ml ConA was used as a polyclonal stimulator for positive control. Evaluation of cytokine production in vitro.