Study On Immune Response Induced By Ppe68Recombinant Bcg In Mice Model

Objective:Construct a shuttle-plasmid pBudCE4.1/PPE68/OriM that the coding sequence of OriM from the plasmid pZM03was subcloned into the Nhel site of the eukaryotic coexpression plasmid pBudCE4.1/PPE68, and then transfected into BCG. BALB/c mice were immunized with PPE68recombinant BCG and the immune response induced by the vaccine was evaluate d.Methods:The OriM gene was amplified by PCR from pZM03and subcloned into Nhel site of the eukaryotic coexpression plasmid pBudCE4.1/PPE68. The shuttle-plasmid was detected by restrictive digestion and DNA sequencing, and then transfected into the murine macrophage cells. The expression of PPE68gene was detected by RT-PCR and Western-blot. The shuttle-plasmid was transformed into BCG and identified by PCR. BALB/c mice were immunized with PPE68recombinant BCG, the DNA vaccine, vector, BCG and saline control groups were set. Two weeks after last immunization, the level of1gG2a, IFN-γ, IL-12and IL-4in the serum of immunized mice was detected, the proliferation response of spleen lymphocyte was measured, CD4+and CD8+T cell percentages were counted, the expression of PPE68recombinant BCG was detected by immunohistochemistry, the numbers of viable bacteria in lung were counted, speens and lungs were prepared for pathological analysis.Results:The expression of PPE68gene in murine macrophage cells was detected by RT-PCR and Western-blot. The PPE68recombinant BCG was successfully constructed. Two weeks after last immunization, compared with BCG, the level of1gG2a in serum were significantly increased after recombinant BCG vaccinated(P<0.05), while the level of IFN-y and IL-12were higher than those of saline control group (P>0.05), but lower than BCG group and DNA vaccined group (P<0.05), and the level of IFN-y and IL-12of DNA vaccined group was the highest, the level of IL-4of each group was not significant. The percentages of CD4+T cells and CD8+T cells in splenolymphocytes of the PPE68recombinant BCG mice were increased, and ratio of CD4+T/CD8+T was decrease.The bacterial colony of lungs can not be observed, the pathological changes of both PPE68recombinant BCG group and BCG group were slightly hyperemia, but the lesions of them were same. There was not obvious pathological changes in the remain lungs and speens Conclusion:The PPE68recombinant BCG was successfully constructed.The vaccine could induce high level of IgG2a, IFN-Y and IL-12in BALB/c mice, strong T cell proliferation response, raised CD4+and CD8+T cell percentages.The PPE68recombinant BCG can induce high level of Thl immune response in mice, and lays a foundation for further research in prevention of tuberculosis.