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目的探讨布氏菌活疫苗皮内注射小鼠产生的体液免疫、细胞免疫应答及免疫保护力。方法将30只小鼠随机分为3组,每组10只,分别为低剂量组(5×107个/只)、高剂量组(2×108个/只)和生理盐水对照组,3组均经后肢皮内注射布氏菌活疫苗,0.1 ml/只。免疫4周后,摘眼球取血,分离血清,制备脾脏淋巴细胞,ELISA法检测小鼠血清中抗Br-PPD Ig G抗体效价;ELISPOT法检测分泌IFNγ、IL-4的脾脏淋巴细胞数;ELISA法检测体外再刺激后小鼠脾脏淋巴细胞分泌的IFNγ水平;流式细胞术对T细胞亚群进行分类。用羊布氏菌M5弱毒株攻击免疫小鼠,通过脾脏荷菌量评价布氏菌活疫苗的免疫保护作用。结果低、高剂量组小鼠血清中抗Br-PPD Ig G抗体效价均较高,几何平均滴度分别为642和557;低、高剂量组小鼠在Br-PPD抗原刺激下,分泌IFNγ、IL-4的脾脏淋巴细胞数以及脾脏淋巴细胞分泌的IFNγ含量,与对照组相比差异均有统计学意义(P均<0.05);CD4+IFNγ+、CD4+IL-4+比例均明显高于对照组,差异均有统计学意义(P均<0.05);低、高剂量组小鼠脾脏荷菌量为0,对照组小鼠为(5.13±0.16)log10 CFU。结论采用皮内注射布氏菌活疫苗的方式免疫小鼠后,能获得较强的体液免疫、细胞免疫应答及免疫保护力。
Objective To investigate the humoral, cellular immune responses and immunoprotective effects of intradermal injection of live Brucella vaccine in mice. Methods Thirty mice were randomly divided into three groups (10 rats in each group): low dose group (5 × 107), high dose group (2 × 108), normal saline control group, All animals were intradermally injected with live Brucella vaccine, 0.1 ml / only. Four weeks after the immunization, blood was taken from the eyeball, and serum was separated to prepare splenic lymphocytes. The anti-Br-PPD Ig G antibody titer was detected by ELISA. The number of spleen lymphocytes secreting IFNγ and IL-4 was measured by ELISPOT. The level of IFNγ secreted by splenic lymphocytes of mice after in vitro restimulation was detected by ELISA. The T cell subsets were classified by flow cytometry. Immunization mice were challenged with the attenuated strain of M. lamblia M5 and the immunological protection of live B. brucei vaccine was evaluated by the amount of spleen load. Results The titer of anti-Br-PPD Ig G in serum of mice in low dose and high dose groups were higher than that of anti-Brd PPD antibody, the geometric mean titers were 642 and 557, respectively. The low and high dose mice secreted IFNγ (P <0.05). The percentage of CD4 + IFNγ + and CD4 + IL-4 + in the spleen lymphocytes were significantly higher than those in the control group (P <0.05). In the low and high dose groups, the spleen load was 0 in the control group and (5.13 ± 0.16) log10 CFU in the control group. Conclusion The mice immunized with intradermal injection of live vaccine against Brucella can acquire strong humoral, cellular immune responses and immune protection.