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目的观察TLR识别配体不同组合佐剂对结核分枝杆菌融合蛋白免疫原性的影响及DDA对TLR识别配体的辅助效应。方法 CpG-ODN(CpG)和/或PolyICLC联合/不联合DDA,分别与融合蛋白Mtb10.4-HspX(MH)混合,制备亚单位疫苗,于第1、4、7周皮下免疫C57BL/6小鼠。以PBS和BCG(仅免疫1次)作为对照。末次免疫后6周,采血检测血清抗体水平,并分离脾淋巴细胞,检测分泌IFN-γ的淋巴细胞水平。结果经MH及HspX抗原刺激后,MH+CpG+PolyICLC+DDA组小鼠分泌IFN-γ的脾淋巴细胞数高于其它各组(P<0.05),MH+CpG+PolyICLC组其次,显著高于MH+CpG及MH+PolyICLC组(P<0.05);联合DDA佐剂组均分别高于对应的未联合DDA佐剂组(P<0.05)。各亚单位疫苗组诱导产生的抗MH及HspX的IgG1、IgG2b、IgG2c水平均明显高于BCG组(P<0.05),其中MH+CpG+PolyICLC+DDA组3种抗体水平最高;各亚单位疫苗组IgG2c/IgG1均高于BCG组(P<0.05)。结论 CpG+PolyICLC+DDA佐剂增强了结核分枝杆菌融合蛋白的免疫原性;CpG和PolyICLC具有佐剂协同作用;DDA有助于CpG和/或PolyICLC诱导特异性细胞免疫应答。
Objective To observe the effect of different combinations of TLR recognition ligands on the immunogenicity of Mycobacterium tuberculosis fusion protein and the adjuvant effect of DDA on TLR recognition ligands. Methods CpG-ODN (CpG) and / or PolyICLC with / without DDA were mixed with the fusion protein Mtb10.4-HspX (MH) respectively to prepare subunit vaccines and subcutaneously immunized C57BL / 6 mice mouse. PBS and BCG (immunized only once) served as controls. Six weeks after the last immunization, blood samples were collected to detect serum antibody levels. Splenic lymphocytes were isolated and the level of IFN-γ-producing lymphocytes was measured. Results The numbers of splenic lymphocytes secreting IFN-γ in MH + CpG + PolyICLC + DDA group were significantly higher than those in other groups (P <0.05), and MH + CpG + PolyICLC group was significantly higher than that in MHC + CpG + PolyICLC group (P <0.05). The combined DDA adjuvant group were higher than the corresponding unadministered DDA adjuvant group (P <0.05). The levels of IgG1, IgG2b and IgG2c of anti-MH and HspX induced by each subunit vaccine group were significantly higher than that of BCG group (P <0.05), among which the three antibodies were the highest in MH + CpG + PolyICLC + DDA group; Group IgG2c / IgG1 were higher than the BCG group (P <0.05). Conclusion CpG + PolyICLC + DDA adjuvant enhances the immunogenicity of Mycobacterium tuberculosis fusion protein; CpG and PolyICLC have adjuvant synergistic effect; DDA can help CpG and / or PolyICLC induce specific cellular immune response.