壳聚糖-海藻酸钠包被的副溶血弧菌外膜蛋白K微球疫苗的制备及其口服免疫效果

Preparation of chitosan-alginate encapsulated microspheres of outer membrane protein K of Vibrio parahaemolyticus and efficiency of oral vaccination

目的制备壳聚糖-海藻酸钠包被的副溶血弧菌外膜蛋白（outer membrane protein,omp）K微球疫苗,并检测其对大黄鱼的口服免疫效果。方法采用乳化-离子交联法制备副溶血弧菌ompK微球疫苗,筛选表面活性剂司盘-80添加量及壳聚糖包被浓度,经正交试验优化微球疫苗的制备工艺,筛选微球疫苗的冻干保护剂。检测采用优化条件制备的微球疫苗的理化特性及其在不同条件下的释放特性,并将微球添加到饵料中,连续5 d投喂大黄鱼,第28天以副溶血弧菌zj2003攻击,检测口服微球疫苗的免疫保护率。结果最适司盘-80添加量为2%～4%,壳聚糖包被浓度为0.6%～0.7%;优化的微球疫苗制备条件为：抗原蛋白浓度10 mg/ml,海藻酸钠浓度1.0%,水油比1∶2,搅拌速度2 000 r/min;微球疫苗的最佳冻干保护剂为2%甘露醇;以优化的条件制备的微球圆整性、分散性好,平均直径为（1.91±1.02）μm,表现出酸性条件稳定、中性和弱碱性条件溶胀的特性;微球疫苗口服免疫的大黄鱼对副溶血弧菌zj2003攻击表现出中等程度的保护力。结论制备了壳聚糖-海藻酸钠包被的副溶血弧菌ompK微球疫苗,并验证了其口服免疫的有效性,为鱼类口服弧菌疫苗的研制及应用奠定了基础。

Objective To prepare chitosan-alginate encapsulated microspheres of outer membrane protein（omp）K of Vibrio parahaemolyticus and determine its immune effect by oral vaccination in large yellow croaker. Methods V. parahaemolyticus ompK microspheres were prepared by emulsification-ionic crosslinking method. Orthogonal experiment of L9（3^4）was used for optimization of parameters for preparation of the microspheres,including the additive amount of Span 80 as a surfactant,the chitosan concentration for coating and the stabilizer. Microspheres prepared under the optimal condition were determined for physicochemical characteristics as well as release under various conditions,then added to the bait,with which large yellow croakers（Pseudosciaena crocea）were fed for 5 consecutive days and challenged with V. parahaemolyticus zj2003 on day 28 after feeding,based on which the protective rate of microspheres was calculated. Results The optimal additive amount of Span 80 was 2% ～ 4%,while the optimal chitosan concentration for coating was 0. 6% ～ 0. 7%. The optimal antigen protein concentration,alginate concentration,ratio of water phase to oil phase,stirring speed and stabilizer were 10 mg / ml,1. 0%,1 ∶ 2,2 000 r / min and 2% mannitol respectively. The microspheres prepared under the optimal condition were globular,at a mean diameter of（1. 91 ± 1. 02）μm,which were well distributed,stable in acidic but disintegrated in neutral and alkaline circumstances. The microspheres by oral vaccination showed a moderate protective effect against challenge with V. parahaemolyticus in large yellow croakers. Conclusion Chitosan-alginate encapsulated microspheres of ompK of V. parahaemolyticus were prepared and verified for efficacy in oral vaccination,which laid a foundation of preparation and application of oral vibrio vaccines for fish use.