EV71 VP1黏膜疫苗制备及其诱导黏膜免疫的实验研究

摘要：目的研究表面展示肠道病毒71型（EV71）结构蛋白VP1的枯草芽孢杆菌芽孢制备疫苗的可行性及应用价值。方法以表面展示EV71 VP1蛋白的重组枯草芽孢杆菌及野生型同基因株枯草芽孢杆菌制备疫苗，并分别通过灌胃＋滴鼻途径免疫BALB／c小鼠（观察组和对照组各16只），4周后摘眼球取血，脱臼处死小鼠收集肺泡及小肠冲洗液，采用ELISA方法检测血清、肺黏膜、肠黏膜中VP1特异性IgA抗体水平。结果枯草芽孢杆菌经培养及溶菌酶处理后均以芽孢形式存在；观察组血清及肺黏膜中VP1特异性IgA抗体水平均显著高于对照组（P均〈0．01），但两组肠黏膜中抗体水平无显著差异。结论展示EV71结构蛋白VP1的枯草芽孢杆菌芽孢可成功制备疫苗，且能有效刺激机体产生特异性黏膜免疫反应；此为EV71疫苗的研制提供了新思路。

Preparation and experimental study of EV71 VP1 mucosa vaccine in mucosal immune response

Objective To study the feasibility and value of bacillus subtilis spores which displayed VP1 of EV71 on surface as vaccine. Methods To prepare vaccines with bacillus subtilis spores displaying VP1 and wild-type isogenic strain, then separately immuned mice orally and intranasally （16 mice in observed group and control group separately）, the eyeballs were picked out to get blood 4 weeks later. The mice were killed by cervical dislocation to collect flushing fluids of pulmonary alveolar and small intestine, then the VP1-specific IgA in serum, lung and intestine were detected by ELISA. Results Bacillus subtilis formed spores after treated with muramidase. VP1-specific IgA in serum and lung of observed group were significantly higher than those of the control group（ both P 〈0.01 ）, but there was no difference of VP1-specific IgA in intestine between the two groups. Conclusion The spores displaying EV71 VP1 can be prepared into vaccine and elicit special mucosal immune response; this study provide a new way in the filed of EV71 vaccine.