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In this study, Mtb8.4 and HspX, which are expressed at proliferating and dormant stages of Mycobacterium tuberculosis (M. tuberculosis), respectively, were chosen to construct two fusion proteins, Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4), and we investigated whether the antigen dose and protein sequential order could impact the immunogenicity and protective efficacy of these fusion protein vaccines against M. tuberculosis. C57BL/6 mice were vaccinated with new constructions containing a fusion protein with adjuvant of N, N′-dimethyl-N, N′-dioctadecylammonium bromide (DDA) or a mixed adjuvant composed of DDA, polyribocytidylic acid and gelatin (DPG), and the antigen specific immune responses and protective efficacy against M. tuberculosis H37Rv were evaluated. The results showed that both antigens, Mtb8.4-HspX and HspX-Mtb8.4, could elicit strong human T cell responses. With the existing of DDA adjuvant, HspX-Mtb8.4 induced significantly higher secretion level of IFN-γ and TNF-α in spleen cells than Mtb8.4-HspX (p < 0.05). In its protective efficacy study, the isolated bacterial Colony Form Unit (CFU) in H8.4-DPG group was significantly reduced compared to 8.4H–DPG group (p < 0.05). Furthermore, with the stimulation of Mtb8.4 in vitro, the secretion of IFN-γ and TNF-α from mice immunized with 20 μg of H8.4 exhibited relative higher level than the group immunized by 7 μg of H8.4 (p < 0.05), whereas, IL-2 secreting showed contrary result. The data suggest that the antigen sequential order and dose selection should be considered when a tuberculosis protein vaccine is to be constructed and its immune strategy is to be planned.We constructed two novel TB subunit vaccine candidate Mtb8.4-HspX and HspX-Mtb8.4.Mtb8.4-HspX and HspX-Mtb8.4 with reversed organization.Mtb8.4-HspX and HspX-Mtb8.4 have different immunogenicity and protective efficacy.