Statistical analysis.  The statistical significance of differential findings between experimental groups was determined by Student’s test. Data were considered statistically significant

at P < 0.05. The recombinant pcDNA3-Ag85A plasmid was confirmed by BamHI and XbaI digestion. The UbGR-Ag85A fusion DNA vaccine was confirmed, respectively, by HindIII and XbaI, BamHI and XbaI, Hind III and XbaI digestion. Finally, the sequences of the two DNA vaccines were shown to be correct by sequencing. After a large scale of preparation, the plasmids were suspended selleck chemicals in endotoxin-free PBS. DNA was quantified by spectrophotometry at 260 nm, and the final concentration of the solution was adjusted to 1 μg/μl of DNA in PBS. The purpose of transfection experiment was to obtain the specific target cells for cytotoxicity assay. After selection by G418 (800 μg/ml), fifteen clones were obtained, and five clones of transfected cells were randomly chosen and screened for Ag85A mRNA by reverse transcription-PCR. After electrophoresis, a specific single band about 1.0 kb in length Akt inhibitor was

observed in clones I, II, III, IV and V. And then, the expression of Ag85A was further examined in clone I by immunocytochemistry. The immunostaining was restricted to the cytoplasm of the cells transfected with pcDNA3-Ag85A plasmid. However, no staining was detected in P815 cells. No staining Adenosine signals were detected with the sera from healthy control people, which indicated that the staining is specific. Those results demonstrated that Ag85A antigen could be expressed stably in P815 cells and that the clone I could be used as target cells in the cytotoxicity assay. To determine the level of Ag85A-specific IgG elicited by different vaccines, mice of different groups were immunized three times at 3-week intervals. Three weeks after the last immunization, the sera from mice were collected by retro-orbital bleeding, and

antigen-specific antibodies were detected by ELISA. As shown in Fig. 1, compared with the pcDNA3 vector group or pcDNA3-ub group, the Ag85A DNA vaccine elicited a significantly higher level of IgG (P < 0.01). However, the IgG level in the UbGR-Ag85A fusion DNA vaccine group was lower than that in Ag85A DNA vaccine group (P < 0.01). The IgG subclasses give an indication of the Th1 versus Th2 nature of the immune response. We also detected the relative ratio of IgG2a/IgG1. As shown in Fig. 2, although the IgG level decreased in the ub fusion DNA vaccine group, the relative ratio of IgG2a/IgG1 increased significantly in the fusion DNA vaccine (P < 0.01), compared with the Ag85A DNA vaccine group. T helper cells play an important role in eliciting both humoral and cellular immune responses via expansion of antigen-stimulated B cells and expansion of CD8+ T cells.