Construction,purified Expression And Immunogenicity Study Of Fusion Protein RS2 In Combination With MnJ Adjuvant

Purpose:Tuberculosis(TB)is an infectious respiratory disease caused by Mycobacterium tuberculosis in patients with predominantly pulmonary lesions.BCG vaccine is currently considered to be the most effective vaccine against TB,but has been shown to have some drawbacks during its long-term use.In view of this,in this study,we propose to construct a fusion protein with the RD region antigen Rv1506c and the secretory phase antigen Rv3204,and stimulate peripheral blood of DIM bferent populations to detect IFN-γsecretion in order to detect its recognition by Mycobacterium tuberculosis(M.tb)-infected T cells.A novel subunit vaccine was constructed by combining a fusion protein with manganese glue adjuvant(MnJ)and immunized with it in C57BL/6 mice to detect the level of immune response and evaluate its immunogenicity.Methods:1、The RD region antigen Rv1506c and the secretory phase antigen Rv3204 were screened and constructed into the fusion protein RS2 by protein Linker,and the constructed fusion protein was verified by SDS-PAGE and Western blot.2.Subject volunteers were recruited from hospitals,and peripheral blood was collected after signing the informed consent form,and the constructed fusion proteins and subfractions were tested for immunogenicity using multiplex microsphere flow immunofluorescence luminescence assay(MFCIA).3.The fusion proteins were mixed with adjuvant and DDA to produce DMI,DMMb and DIMb subunit vaccines,and mice were immunized with the vaccination protocols of 0,10,7,7 and 7 days.The Ig G antibody titers in mouse serum,the changes in the levels of various cytokines in mouse splenocyte cultures and the DIM bferent T cell subpopulations in splenocytes were detected by flow cytometry(FCM).in splenocytes.Results:1.The fusion protein RS2 was successfully constructed,and the endotoxin level was controlled at 0.1 EC/m L as verified by SDS-PAGE and WB.2.The fusion protein and subcomponent proteins stimulated the peripheral blood of the population and induced the secretion of higher levels of IFN-γ,IL-2,IL-6 and TNF-α.3,The subunit vaccines RS2+DMI,RS2+DMMB and RS2+DIMB were successfully prepared and immunized in C57BL/6 mice.It was found that after the expected immunization time,high titers of total Ig G were induced in the peripheral blood serum of mice,and the titers of Ig G1 as well as Ig G2a also showed a significant increase,and the highest titers of total Ig G antibodies and their subclasses were secreted in the BCG+RS2+DMI group.4.The secretion levels of IFN-γ,TNF-αand IL-2,which are Th1-type cytokines,in the supernatant cultures of splenocytes from mice in the primary immune enhancement group were significantly increased,especially in the BCG+RS2+DMI group and BCG+RS2+DMMB group,which were much higher than the other groups.5.The results of various splenocytes and subpopulations of splenocytes detected by FCM indicated that the number of both IFN-γ~+CD4~+T single-positive cells,TNF-α~+CD4~+T single-positive cells and IFN-γ~+TNF-α~+CD4~+T double-positive cells were significantly higher than those of other groups,and among all groups,especially the BCG+RS2+DMI group was the highest.Conclusion:1,The fusion protein RS2 was successfully designed,constructed and expressed,and both SDS-PAGE and WB were verified as expected.2.The immunogenicity of the fusion protein RS2 and subcomponent proteins was confirmed by MFCIA assay.The results of the population assay showed that the fusion protein RS2 was strongly immunogenic and could be recognized by peripheral blood T cells of M.tb-infected patients,stimulating the secretion of high levels of Th1-type cytokines in peripheral blood of DIM bferent populations.3,RS2 combined with DIM bferent adjuvants immunized mice induced high titers of Ig G-like antibodies in the serum;the secretion levels of CD4~+Th1-type cytokines IFN-γ,TNF-αand IL-2 were also significantly increased;FCM detected a significant increase in the number of IFN-γ+/IL-2+single-positive and double-positive CD4~+T cells.The above results suggest that the new subunit vaccine based on RS2 combined with manganese gum adjuvant has strong immunogenicity,which may be beneficial to the short-term anti-M.tb infection effect and thus make up for the shortcomings of BCG protection.4,After RS2 combined with DIM bferent adjuvant combinations(DMI,DMMb,DIMb)immunized mice,the vaccine group containing MnJ adjuvant(DMI,DMMb)could provide stronger immunogenicity.5.This study provides data validation for the construction of fusion proteins in subunit vaccines and the validation of the primary immunization enhancement strategy,in addition,it provides an experimental basis for the combination strategy of fusion proteins with compound adjuvant subunit vaccines.Figure[11]Table[9]Reference[57]...