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目的探讨结核分枝杆菌融合蛋白亚单位疫苗对BCG初始免疫的加强效应及保护效力。方法将Ag85B、Ag85B-Mpt64190-198-HspX(AMH)、Ag85B-Mpt64190-198-Mtb8.4(AMM)以及AMH+AMM蛋白分别与佐剂二甲基三十六烷基铵(DDA)和卡介苗多糖核酸(BCG-PSN)混合,制备亚单位疫苗。第0周用BCG皮下免疫C57BL/6小鼠,第8、10周分别用各蛋白疫苗皮下加强免疫,以PBS和BCG(仅免疫1次)作为对照。末次免疫后4周,采血检测血清抗体水平,并分离脾淋巴细胞,检测分泌IFNγ的水平;末次免疫后12周,经尾静脉注射H37Rv株,6周后取肺,进行菌落计数,抗酸及HE染色观察。结果各亚单位疫苗加强组诱导产生的特异性抗Ag85BIgG抗体水平均明显高于BCG组;融合蛋白疫苗加强组免疫小鼠脾细胞经Ag85B和PPD刺激后,产生分泌IFNγ的淋巴细胞数明显多于BCG组;融合蛋白加强组免疫小鼠肺组织结核结节面积与BCG组比较,差异无统计学意义;仅AMM+AMH加强组免疫小鼠肺组织菌落计数明显低于BCG组,其抗酸染色阳性细菌数明显低于PBS、BCG及AMH、Ag85B加强组。结论AMH和AMM疫苗联合加强BCG免疫,能诱导小鼠特异性的细胞免疫和体液免疫应答,具有较强的免疫原性,可增强BCG的保护效力。
Objective To investigate the enhancing effect and protective effect of Mycobacterium tuberculosis fusion protein subunit vaccine on BCG immunization. Methods Ag85B, Ag85B-Mpt64190-198-HspX (AMH), Ag85B-Mpt64190-198-Mtb8.4 (AMM) and AMH + AMM proteins were incubated with adjuvant dimethyltriacontamines (DDA) and BCG Polysaccharide nucleic acids (BCG-PSN) were mixed to prepare subunit vaccines. At week 0, C57BL / 6 mice were immunized subcutaneously with BCG, and were immunized subcutaneously with each protein vaccine at weeks 8 and 10, respectively. PBS and BCG (immunized only once) were used as controls. Four weeks after the last immunization, blood samples were collected to detect serum antibody levels, and splenic lymphocytes were isolated to detect the level of secreted IFNγ. At 12 weeks after the last immunization, H37Rv strain was injected through tail vein and lungs were taken after 6 weeks for colony counting, HE staining observation. Results The levels of specific anti-Ag85B IgG antibodies induced by each subunit vaccine-boosting group were significantly higher than those of BCG group. The number of lymphocytes secreting IFN-γ secreted by splenocytes of mice immunized with the fusion protein vaccine was significantly higher than that of the group stimulated by Ag85B and PPD BCG group; the area of tuberculosis nodules in lung tissue of mice immunized with fusion protein was not significantly different from that of BCG group; the colonies count of lung tissue of mice immunized with AMM + AMH alone group was significantly lower than that of BCG group The number of positive bacteria was significantly lower than PBS, BCG and AMH, Ag85B enhanced group. Conclusion AMH and AMM combined with BCG immunization can induce specific cellular and humoral immune responses in mice with strong immunogenicity and enhance the protective effect of BCG.