渔用弧菌疫苗注射佐剂的筛选和口服微粒的研制

发布时间：2019-04-24 00:26

【摘要】： 本文选用铝胶佐剂和白油佐剂为弧菌疫苗注射用佐剂,制备了两种佐剂苗和一种水剂苗腹腔注射免疫红笛鲷,用间接ELISA法测定血清抗体滴度,并对其免疫保护率进行了评价。结果表明,红笛鲷在免疫后都发生特异性免疫反应,所有免疫组与对照组间抗体效价都存在显著差异(P0.01),铝胶组提升抗体效价比较快下降也比较快,白油佐剂组抗体提升后能够维持相对较长的一段时间。56天后各组分别用溶藻弧菌和哈氏弧菌攻毒,白油佐剂组免疫保护率为85.4%和82.6%,铝胶佐剂组为47.9%和41.3%,而水剂组为16.7%和21.7%。本文选用海藻酸钠作为口服投递载体,采用乳化复沉淀方法包裹哈维氏弧菌全菌制备成疫苗微球,并对微球的制备工艺进行了优化,结果表明:3%(m/v)海藻酸钠溶液,菌浓度4×109 CFU. mL-1,水油相比1:9(0.1%Span80乳化剂),转速2000 r/min为其最佳制备工艺,微球疫苗粒径为37.5±0.478μm,跨度0.46,微球包封率高达98.6%。微球在体外模拟胃液和肠液12h释放率分别为10.2%和9.3%,24h释放率分别为16.8%和13.2%。FITC标记的荧光微球示踪表明微球能通过消化道到达后肠,1-24h在后肠能够检测到荧光微球,并可以明显看到后肠对荧光微球的吸收。免疫组织化学结果显示后肠在灌胃1天后免疫组化能检测到棕黄色的阳性颗粒,只到第7天还能在后肠检测到。微球疫苗口服免疫红笛鲷后,用间接ELISA法测定血清、肠粘液和体表粘液抗体效价,结果表明,红笛鲷在免疫后都发生特异免疫反应,灌胃组和投喂组在第四周时血清抗体效价都达到峰值1:4096和1:2048;肠粘液抗体效价在第3周和第5周分别达到峰值1:1024和1:512;体表粘液抗体效价和对照组没有明显变化。免疫30天后用活菌攻毒,测定受免鱼的免疫保护率,口服微粒灌胃组和投喂组30天后的免疫保护率分别为63.1%和47.3%。综合以上结果表明海藻酸钠能作为弧菌口服疫苗的投递载体,微球疫苗具有较好的免疫效果。
[Abstract]:In this paper, aluminum gel adjuvant and white oil adjuvant were used as adjuvant for injection of Vibrio vaccine. Two kinds of adjuvant vaccine and one kind of admixture vaccine were prepared by intraperitoneal injection of red sea bream. The titers of serum antibody were determined by indirect ELISA method, and the immune protection rate was evaluated. The results showed that the specific immune reaction occurred after immunization, and the antibody titers of all immunization groups were significantly different from those of control group (P0.01). The antibody titer of aluminum gel group increased rapidly and decreased rapidly. The results showed that the antibody titers of red sea bream were significantly different from those of control group (P0.01). After 56 days of challenge, Vibrio alginolyticus and Vibrio harringensis were used in each group, and the immune protective rates were 85.4% and 82.6% in the white oil adjuvant group, and the protective rates were 85.4% and 82.6% in the white oil adjuvant group, respectively. It was 47.9% and 41.3% in aluminum adjuvant group and 16.7% and 21.7% in water group. In this paper, sodium alginate was used as an oral delivery carrier to prepare vaccine microspheres by encapsulating whole Vibrio harvestris with emulsified precipitation method. The preparation technology of the microspheres was optimized. The results showed that 3% (mv) sodium alginate solution was used to prepare vaccine microspheres, and the results were as follows: 3% (mv) sodium alginate solution, Bacterial concentration 4 脳 109 CFU. ML-1, water-oil ratio was 1:9 (0.1%Span80 emulsifier) and rotating speed was 2000 r/min. The particle size of the microsphere vaccine was 37.5 卤0.478 渭 m, the span was 0.46, and the encapsulation efficiency of the microspheres was up to 98.6%. The release rates of microspheres in simulated gastric juice and intestinal fluid were 10.2% and 9.3% in vitro, and 16.8% in 24h and 16.8% in 24 hours, respectively. The fluorescent microspheres labeled with 13.2%.FITC showed that the microspheres could reach the hindgut through the digestive tract. The fluorescent microspheres could be detected in the hindgut for 24 h, and the absorption of the fluorescent microspheres could be seen obviously in the hindgut. The immunohistochemical results showed that brown positive granules could be detected in the hindgut one day after gavage, but only in the hindgut on the 7th day. The antibody titers of serum, intestinal mucus and body surface mucus were determined by indirect ELISA after oral immunization of red sea bream with microsphere vaccine. The results showed that red sea bream all had specific immune reaction after immunization. At the fourth week, the titers of serum antibody in the gastric and feeding groups reached the peak values of 1? 4096 and 1? 2048 respectively. The antibody titers of intestinal mucus reached the peak values of 1? 1024 and 1? 512 at the 3rd and 5th week, respectively, while the antibody titers of the body surface mucus did not change significantly as compared with that of the control group. After 30 days of immunization with live bacteria, the immune protection rate of the immunized fish was determined. After 30 days of oral administration, the immune protection rates of the two groups were 63.1% and 47.3%, respectively, and that of the control group was 63.1% and 47.3%, respectively. The above results indicated that sodium alginate could be used as delivery carrier for oral vaccine of Vibrio, and the microsphere vaccine had better immune effect.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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