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目的研究聚-DL-乳酸-聚乙二醇共聚物(DL-polylactide-co-polyethylene glycol,PELA)包裹哈维弧菌(Vibrio har-veyi,Vh)重组外膜蛋白OmpK制备的微球疫苗的部分特性及其对鲫鱼(Carassius auratus gibelio)的口服免疫效果。方法用可生物降解的合成高分子材料PELA,通过乳化溶剂挥发法包裹OmpK,制备微球疫苗,Bradford法测定蛋白浓度,计算蛋白包裹率;通过扫描电镜观察微球粒径大小及其在4℃保存不同时间的形貌。取鲫鱼随机分为3组:微球疫苗组、OmpK蛋白组和空白对照组,前两组采用疫苗或蛋白拌饲投喂方式口服免疫,投喂3 d,间隔7 d,再投喂3 d加强免疫;对照组投喂不含疫苗的饲料。加强免疫4周和8周后,经腹腔注射20 LD50的Vh EcGS020802株攻击,记录各组鱼死亡数,计算相对免疫保护率。结果制备的PELA-OmpK微球疫苗中OmpK蛋白的包裹率达78%。微球粒径小于12μm,且90%微球的粒径小于5μm。4℃保存10 d微球表面光滑、圆整;保存30 d PELA微球降解,粒径较大的微球表面出现空洞;保存60 d可见大量碎片,微球表面毛糙松散,出现多个空洞。微球疫苗加强免疫鲫鱼4周和8周后,对活菌攻击的相对免疫保护率分别为70%和48%,而口服OmpK蛋白组和空白对照组鱼全部死亡。结论用PELA包裹裸疫苗制备成微球疫苗,对抗原具有一定的保护作用,PELA用作鱼类口服疫苗的投递载体是可行的。
OBJECTIVE: To study the effect of microspheres vaccine prepared by encapsulating Vibrio har-veyi (Vh) recombinant OMPK in DL-polylactide-co-polyethylene glycol (PELA) Partial Characteristics and Its Oral Immune Effect on Carassius auratus gibelio. Methods The biodegradable synthetic polymer material PELA was used to encapsulate OmpK by emulsion solvent evaporation method to prepare microspheres vaccine. The protein concentration was determined by Bradford method and the protein inclusion rate was calculated. The size of the microspheres and the size of the microspheres at 4 ℃ Save the appearance of different time. The crucian carp were randomly divided into three groups: microspheres vaccine group, OmpK protein group and blank control group. The first two groups were immunized orally with the vaccine or protein mixed feeding and feeding method for 3 d Strengthen immunity; control group fed without vaccine feed. After 4 and 8 weeks of booster immunization, 20 LD50 Vh EcGS020802 strain was intraperitoneally challenged. The number of fish deaths in each group was recorded and the relative immune protection rate was calculated. Results The encapsulation efficiency of OmpK protein in PELA-OmpK microsphere vaccine was 78%. The size of the microspheres is less than 12 μm, and the diameter of the 90% microspheres is less than 5 μm. The surface of microspheres preserved at 4 ℃ for 10 days was smooth and rounded. The PELA microspheres degraded 30 days after storage, and the microspheres with larger particle size showed voids on the surface. Large amounts of debris were observed after 60 days storage, and the microspheres were rough and loose with many voids. The immunization protection rate of viable bacteria was 70% and 48% respectively after the microspheres vaccine boosted the crucian carp four weeks and eight weeks later, while the OmpK protein group and the blank control group all died. Conclusions The microspheres vaccine prepared by PELA-encapsulated naked vaccine has certain protective effect on the antigen. It is feasible that PELA is used as delivery vector for oral vaccines of fish.