Prokaryotic expression and immunogenicity assessment of Mycobacterium tuberculosis Rv2005
Objective:To select advantageous target antigens of vaccine for new tuberculosis(TB)by constructing the Myco-bacterium(M.tuberculosis)Rv2005 carrier,and assessing prokaryotic expression and immunogenicity.Methods:The specific primers of the target gene Rv2005 were designed and amplified by PCR.The recombinant plasmid pET28a-Rv2005 was construct-ed by molecular cloning technique,expressed prokaryotically,and purified by nickel column affinity chromatography.Peripheral blood of M.tuberculosis infected persons and healthy controls(HCs)was stimulated with purified rRv2005,and the corresponding levels of specific IFN-γ antibodies produced by peripheral blood T lymphocytes were detected by multiple microsphere flow immu-nofluorescence assay(MFCIA).A compound liposomal adjuvant MC based on TLR4/9 agonist monophosphoryl lipid A(MPLA)/oligodeoxynucleotide sequence containing non-methylated guanine and cytosine dinucleic acid(CPGODN)was con-structed,which was combined with rRv2005 to immunize C57BL/6 mice,and killed after 9 weeks.Enzyme-linked immunosor-bent assay(ELISA)was used to detect the titers of specific antigens IgG,IgG1,and IgG2a in the serum of immunized mice and the secretion levels of specific antigens Th1 type cytokines IFN-γ,TNF-α,and IL-2 in the supernatant of spleen cells of immu-nized mice.Results:The recombinant plasmid pET28a-Rv2005 was successfully constructed,and its expression was identified by SDS-PAGE and Western blot.After rRv2005 stimulation,compared to active tuberculosis(ATB)and latent tuberculosis infec-tion(LTBI)subjects,the levels of cytokines released by T lymphocytes in peripheral blood of HCs were significantly increased,and the levels of cytokines secreted by ATB subjects were the highest(P<0.01).The mice immunized with BCG and rRv2005 combined with MC produced higher levels of IgG and its subclasses,and the IgG2a/IgG1 ratio was>1,which tended to be Th1 type cell immunity.After PPD and rRv2005 stimulation,the spleen cells in the BCG initial immune enhancement group can induce the release of high levels of antigen-specific IFN-γ,TNF-α and IL-2.Conclusion:rRv2005 can induce the release of high levels of IFN-γ from peripheral blood T lymphocytes of Chinese TB patients.rRv2005 combined with MC can stimulate Th1 cell immunity in mice.BCG initial immunity and rRv2005/MC enhancement may effectively compensate for the deficiency of BCG protection.The complex adjuvant MC based on TLR agonist has a good application prospect.
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