Objective To investigate the effects of Al(OH)3 and CpG oligodeoxynucleoties adjuvants on the immunogenici-ty of Mycobacterium tuberculosis(Mtb)Ag85B protein.Methods Ag85B protein of Mycobacterium tuberculosis was ex-pressed in E.coli and purified by affinity chromatography.Then,BALB/c mice were immunized subcutaneously with the pu-rified Ag85B protein formulated with Al(OH)3,CpG oligodeoxynucleoties(CpG for short),or compound adjuvant of Al(OH)3 and CpG(Al(OH)3+CpG)respectively and booster immunization 28 days later.Serum samples were collected at 14,42 and 56 days after the primary vaccination.ELISA was used to detect the levels of Ag85B-specific IgG,lgG1,IgG2a and IgG2b.Forty-two days after primary immunization,spleen lymphocytes were aseptically isolated from 4 mice in each group.The number of interferon-γ(IFN-γ)and interleukin-4(IL-4)secreting lymphocytes in each group was detected by enzyme-linked immunosorbent spot,and the proportion of CD4+and CD8+T lymphocytes in each group was detected by flow cy-tometry.Results High levels of Ag85B-specific IgG were in-duced by immunization with 1.5 μg or 4.5 μg of recombinant Ag85B protein formulated with Al(OH)3+CpG,which was better than the protein formulated with either adjuvant alone.The IgG subclasses in mice immunized with 1.5 μg or 4.5 μg recombinant Ag85B protein formulated with Al(OH)3+CpG were IgG1,IgG2a and IgG2b,and the levels of IgG2a and IgG2b were higher than those of the same protein formulated with Al(OH)3,exhibiting statistically significant difference(all P<0.001).Enzyme-linked immunosorbent spot showed that the number of spleen lymphocytes secreting IFN-γ and IL-4 in mice immunized with 4.5 μg recombinant Ag85B protein with Al(OH)3+CpG was higher than those of the same protein formulated with Al(OH)3,exhibiting statistically significant differ-ence(both P<0.05).Flow cytometry showed that the proportion of CD4+T lymphocytes in T lymphocytes of mice immu-nized with 1.5 μg or 4.5 μg recombinant Ag85B protein formulated with Al(OH)3+CpG was higher than that of the same protein formulated with Al(OH)3.Conclusion The combination of Al(OH)3 and CpG may enhance the immunogenicity of recombinant Ag85B protein.
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