壳聚糖-海藻酸盐复合微囊疫苗的制备及其对斜带石斑鱼的口服免疫效果  

Preparation and immune effect of chitosan-alginate microcapsules containing Vibrio harveyi antigen OmpK in orange-spotted groupers

作　　者：任燕[1] 张小江[1] 陶家发[1] 李宁求[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业部渔药创制重点实验室,广东广州510380

出　　处：《中国生物制品学杂志》2015年第2期110-114,共5页Chinese Journal of Biologicals

基　　金：国家"863"项目(2006AA100308);广东省科技计划(2012A020601006)

摘　　要：目的制备包裹哈维弧菌（Vibrio harveyi,Vh）重组外膜蛋白Omp K的壳聚糖-海藻酸钠复合微囊疫苗,并检测其对斜带石斑鱼的口服免疫效果。方法采用乳化-复乳化法将壳聚糖、海藻酸钠包裹哈维弧菌重组外膜蛋白Omp K,制备微囊疫苗,测定其粒径、包裹率及载抗原量;经口灌服免疫斜带石斑鱼,2周后进行加强免疫,设Omp K蛋白组和空白对照组,ELISA法检测免疫后斜带石斑鱼血清及后肠黏膜上清抗体水平;于免疫后30 d,经斜带石斑鱼腹腔注射1.0×10^7 CFU/ml的哈维弧菌,计算各组相对保护率（relative percent survival,RPS）。结果微囊为球形或类球形,粒径5.0~20.0μm,平均粒径10.6μm;微囊中蛋白平均包裹率为71.26%,平均载抗原量为1.67%。微囊疫苗组血清抗体效价峰值为29,明显高于Omp K蛋白组（P〈0.01）;微囊疫苗组后肠黏膜上清的吸光值（A450-A630）明显高于Omp K蛋白组和空白对照组（P〈0.01）。微囊疫苗组的RPS达62.5%,高于Omp K蛋白组（P〈0.01）。结论制备的壳聚糖-海藻酸盐复合微囊疫苗口服石斑鱼后,具有较好的免疫效果,作为鱼类口服疫苗的投递载体是可行的。Objective To prepare chitosan-alginate microcapsules containing the outer membrane protein（Omp K） of Vibrio harveyi and determine its immune effect in orange- spotted groupers by oral route. Methods The Omp K of V. harveyi was entrapped with chitosan-alginate microcapsules by emulsification-reemusification method,and the prepared microcapsules were determined for size,entrapment rate and antigen loading. Orange-spotted groupers were immunized twice with the prepared microcapsules by oral route at an interval of 2 weeks,while Omp K and blank controls were set up. The antibody levels in sera and intestine mucosa supernatant were determined by ELISA. The orange-spotted groupers were challenged i. p. with 1. 0 × 10^7 CFU / ml of V. harveyi 30 d after immunization,and the relative percent survivals（RPSs） of various groups were calculated. Results The chitosan-alginate microcapsules were in spherical shape at diameters of 5. 0 ~ 20. 0 μm,with a mean of 10. 6 μm. The mean encapusulation efficiency and loading percent of microspheres were 71. 26% and 1. 67% respectively. The maximum antibody titer in sera of orange-spotted groupers immunized with the prepared microcapsules was 29,which was significantly higher than that in Omp K group（P 〈 0. 01）,while the absorbance（A450-A630）of hindgut mucus was higher than those in Omp K and blank control groups（P 〈 0. 01）,and the RPS（62. 5%） was higher than that in Omp K control group（P 〈 0. 01）. Conclusion The prepared chitosanalginate microcapsules showed good immune effect in orange-spotted groupers,which was feasible to be used as a delivery carrier of oral vaccine for fish.

关 键 词：壳聚糖 海藻酸盐 外膜蛋白 微囊 疫苗 斜带石斑鱼 口服免疫 

分 类 号：S942.5[农业科学—水产养殖]