Résumé
@#Objective To evaluate the immunogenicity and protective effect of Mycobacterium tuberculosis(M.tb) Ag85B-Fc2a DNA vaccine in mice,so as to provide experimental basis for the development of the vaccine.Methods The recombinant plasmid pcD-Ag85B-Fc2a was identified by double digestion and sequencing,and then transfected into CHO-K1 cells.The expression of fusion protein Ag85B-Fc2a was detected by Western blot.Vector pcDNA3.1(+) and recombinant plasmid pcDAg85B-Fc2a were injected into female C57BL/6J mice through thigh muscle respectively(control group and immunization group),with 10 mice in each group,and booster immunization was carried out two weeks after the initial immunization.At14 d,28 d and 42 d after the initial immunization,serum samples was separated,and the titers of IgG antibody in the serum were detected by indirect ELISA.At 42 d after the initial immunization,the spleen of mice was taken aseptically and made into single cell suspension,and the proportions of CD4~+and CD8~+T cells were detected by flow cytometry.The remaining mice were injected with M.tb H37Ra through the tail vein 42 d after the initial immunization at a dose of 10~6 CFU/mouse.After 28 d of challenge,the lung and spleen of mice were collected aseptically.The number of bacteria in the left lung and spleen was measured by plate method,and the bacteria in the right lung was detected by auramine O fluorescence staining.Results Double digestion and sequencing results showed that the recombinant plasmid pcD-Ag85B-Fc2a was constructed correctly.After transfection into CHO-K1 cells,the fusion protein Ag85B-Fc2a with a relative molecular mass of about 70 000was detected.The Ag85B-specific IgG antibody titer in serum of mice in the immunization group was 1:3 200,1:12 160,and 1:12 800 at 14 d,28 d,and 42 d after the initial immunization,respectively,but no antibody titer was detected in the serum samples of control group.At 42 d after the initial immunization,the percentages of CD4~+ T cells in mouse spleen of control group and immunization group were(23.61±0.64)% and(26.92±0.80)%,and the percentages of CD8~+T cells were(14.12±0.87)% and(18.78±0.94)%,respectively,with significant differences(t=3.23 and 3.64,respectively,each P <0.05).After infection with M.tb H37Ra for 28 d,the numbers of bacteria were(4.73±0.13) and(3.81±0.14)CFU in the left lung,and(5.02±0.19) and(4.30±0.13) C.FU in the spleen of control group and immunization group,respectively,with significant differences(t=4.65 and 3.12,respectively,each P <0.01).The bacteria loading in the right lung was consistent with that in the left lung.Conclusion Ag85B-Fc2a DNA vaccine can induce specific humoral and cellular immune effects in mice,and can produce good protective effect against M.tb H37Ra infection.