布氏菌活疫苗滴鼻免疫小鼠的免疫学评价

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目的评价布氏菌活疫苗滴鼻免疫小鼠后黏膜、体液及细胞免疫的效果。方法将60只小鼠随机分为低剂量组(5×107个/只)、高剂量组(3×108个/只)和阴性对照组(生理盐水),均于小鼠清醒状态下进行滴鼻免疫布氏菌活疫苗,剂量为10μl/只(5μl/鼻孔)。免疫30 d后,分离血清,制备脾脏淋巴细胞及肺泡灌洗液。间接ELISA法检测免疫小鼠血清中Ig G抗体效价、肺泡灌洗液中Ig G、Ig A抗体效价及体外再刺激后小鼠脾细胞分泌IFNγ、IL-4水平;ELISPOT法检测分泌IFNγ、IL-4的脾脏淋巴细胞数目;流式细胞术检测T细胞亚群分类。用羊布氏菌弱毒株M5经皮下和滴鼻两种途径攻击免疫小鼠,通过脾脏细菌计数评价布氏菌活疫苗的免疫保护作用。结果低剂量组和高剂量组小鼠血清中Ig G抗体效价及小鼠肺泡灌洗液中的Ig G、Ig A差异无统计学意义(P>0.05);低剂量组和高剂量组小鼠在Br-PPD抗原和灭活菌体抗原刺激下,分泌IFNγ的脾脏淋巴细胞数、体外再刺激后小鼠脾细胞分泌IFNγ的水平及脾淋巴细胞中CD4~+IFNγ~+细胞比例均明显高于阴性对照组(P<0.05);攻毒试验结果表明,布氏菌活疫苗具有良好的保护力。结论布氏菌活疫苗经滴鼻免疫小鼠后可获得良好的免疫效果和保护力。 Objective To evaluate the effect of mucosal, humoral and cellular immunity after intranasal immunization with live Brucella vaccine. Methods Sixty mice were randomly divided into low dose group (5 × 107 cells / group), high dose group (3 × 108 cells / group) and negative control group (saline) Nasal immunization with live Brucella vaccine at a dose of 10 μl / mouse (5 μl / nostril). After 30 days of immunization, serum was separated to prepare spleen lymphocytes and alveolar lavage fluid. Indirect ELISA was used to detect Ig G antibody titers in serum, Ig G and Ig A antibody in BALF and levels of IFNγ and IL-4 secreted by mouse splenocytes after in vitro stimulation. ELISA was used to detect the secretion of IFNγ , The number of spleen lymphocytes of IL-4, and the classification of T cell subsets by flow cytometry. Immunized mice were challenged with M. lamblia strain M5 via subcutaneous and intranasal routes. The spleen bacterial count was used to evaluate the immunoprotection of live B. brucei vaccine. Results There was no significant difference in Ig G antibody titers between mice in low dose and high dose groups and Ig G and Ig A in mouse alveolar lavage fluid (P> 0.05). Low dose group and high dose group were smaller The numbers of splenic lymphocytes secreting IFNγ stimulated by Br-PPD antigen and inactivated bacterial antigen were significantly higher than those of splenic lymphocytes stimulated by Br-PPD antigen and IFN-γ Higher than the negative control group (P <0.05). The results of the challenge test showed that the live vaccine of Brucella has good protection. Conclusion The live vaccine of Brucella can improve the immunity and protect the mice after intranasal immunization.
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