| Objective:To construct a fusion protein SHR3 containing multi-stage antigens during the growth period of M.tb,to dectect whether SHR3 and its sub-component proteins could be recognized by peripheral blood T lymphocytes of M.tb infected subjects in Huainan City,Anhui Province,to immunize C57BL/6 mice with SHR3 in combination with adjuvant DMT and to evaluate its immunogenicity.Methods:1.Predominant antigens expressing during stage of secretory phase(Rv1626),incubation period(Rv2866),and resuscitation period(Rv1884)after.tb infection were selected respectively.Prokaryotic expression vectors of the fusion protein SHR3 and its sub-component proteins were constructed,prokaryotic expressed,purified,and identified by SDS-PAGE and Western-blotting.2.All subjects were selected according to the clinical diagnostic criteria for TB and divided into infected patients group(including TB patients and latent infections)and healthy controls group.Whole-blood IFN-? release assay(Whole-blood IFN-? release assay,WBIA)was performed to detect the levels of specific IFN-? induced by SHR3 and its sub-component proteins.Meanwhile,SHR3 specific CD4+ and CD8+ T cells which secret for single-positive and double-positive cytokines(IFN-?+ and IL-2+)were detected and analyzed by flow cytometry and ICS.3.SHR3/DMT were constructed,C57BL/6 mice were divided into PBS negative control group,DMT group,BCG positive control group,SHR3/DMT group and BCG+SHR3/DMT group.Each group of C57BL/6 mice were immunized subcutaneously according to the corresponding immunization protocol.After 9 weeks,the mice were sacrificed and SHR3-specific antibody titer,IL-4,IL-2,TNF-? and IFN-? levels secreted by the splenic lymphocytes were detceted by ELISA.The mRNA expression levels of IFN-??TNF-? and iNOS from the lung were also detected by qRT-PCR.Results:1.The fusion protein SHR3 and its sub-component proteins were constructed,expressed,purified successfully,and confirmed by SDS-PAGE and Western-blotting.2.SHR3 and its sub-component proteins induced the statistically higher level of IFN-? stimulated by lymphocytes from subjects of M.tb-infected than the healthy control,as well as the numbers of CD4+ T and CD8+ T cells which secret for single-positive and double-positive cytokines(IFN-?+ and IL-2+).3.SHR3/DMT induced significant higher levels of specific IgG,Ig2a and IgG1 antibodies(IgG2a/IgG1 indicated that SHR3/DMT mainly induced the Thl-type cell-mediated immune response),also the mRNA expression levels of IFN-?,TNF-?and iNOS from the lung than those of PBS group ad DMT group.Moreover,whether stimulated by PPD or SHR3,splenic lymphocytes from BCG+SHR3/DMT mice induced the highest levels of IL-2,IFN-? and TNF-?,which were significantly higher than those in PBS group and DMT group(P<0.05).Conclusion:The fusion protein SHR3 consisting of multi-stage target antigens was constructed,which could be well recognized by T cells of.tb infected subjects from Huainan City,Anhui Province.SHR3 could induce higher levels of IFN-y in peripheral blood lymphocytes from M.tb infected patients and more CD4+T and CD8+T cells which secret for single-positive and double-positive cytokines(IFN-?+ and IL-2+).After imnunized,SHR3/DMT induced statistically higher levels of specific IFN-?,TNF-?and IL-2 from splenic lymphocytes.In conclusion,SHP could induce antigen-specific CD4+Th1 type response.This research established a research basis for further protective evaluation of SHR3 against M.tb infection in animal models.Figure[15]Table[8]Reference[57]... |
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