Expression of phosphoenolpyruvate carboxykinase of Mycobacterium bovis BCG and relative immune responses in mice

ObjectiveTo express the recombinant protein of phosphoenolpy ruvate carboxykina se(PEPCK)of Mycobacterium bovis and research the protective immune responses induced by the protein.MethodsThe recom binant plasmid of pHF/pckA which included pckA gene of Mycobacterium bovis was transformed into Escherichia coli HB₁₀₁(E.coli HB₁₀₁).The recombinant protein of PEPCK was expressed and identified by SDS-PAG Eand Western blot.Sixty BALB/c mice were divided into two groups.Each mouse in experimental group was injected with emulsion containing 10μg PEPCK antigen(Ag)in PBS and equal volumes of incomplete Freunds adjuvant(Difco)through the peritoneal.In control group, each mouse was only injected with equal volumes of incomplete Freunds adjuvant.All mice were mimunized for three times at an interval of two weeks.Spleens and blood were taken for the detection of CD4⁺,CD8⁺ T cells and cytokines 2 weeks after the final mimunization.ResultsThe recombinant PEPCK was expressed and identified by Western blot.The spleen of mouse in PEPCK mimunized group was much bigger than that of control group.The number of CD4⁺ T cells and the ratio of CD4⁺/CD8⁺ in the PEPCK mimunized group were significantly higher than that of control group(P<0.01).The amount of interferon gama,inter leuk in 12 and tumor necrosis factor alpha in PEPCK immunized mice was higher than that of control group(P<0.05).ConclusionPEPCK can induce strong cellular and humoral mimune responses and may be a good cand idate for TB vaccine.