漁用弧菌疫苗注射佐劑的篩選和口服微粒的研制
發(fā)布時間：2019-04-24 00:26
【摘要】： 本文選用鋁膠佐劑和白油佐劑為弧菌疫苗注射用佐劑,制備了兩種佐劑苗和一種水劑苗腹腔注射免疫紅笛鯛,用間接ELISA法測定血清抗體滴度,并對其免疫保護率進行了評價。結(jié)果表明,紅笛鯛在免疫后都發(fā)生特異性免疫反應(yīng),所有免疫組與對照組間抗體效價都存在顯著差異(P0.01),鋁膠組提升抗體效價比較快下降也比較快,白油佐劑組抗體提升后能夠維持相對較長的一段時間。56天后各組分別用溶藻弧菌和哈氏弧菌攻毒,白油佐劑組免疫保護率為85.4%和82.6%,鋁膠佐劑組為47.9%和41.3%,而水劑組為16.7%和21.7%。 本文選用海藻酸鈉作為口服投遞載體,采用乳化復(fù)沉淀方法包裹哈維氏弧菌全菌制備成疫苗微球,并對微球的制備工藝進行了優(yōu)化,結(jié)果表明:3%(m/v)海藻酸鈉溶液,菌濃度4×109 CFU. mL-1,水油相比1:9(0.1%Span80乳化劑),轉(zhuǎn)速2000 r/min為其最佳制備工藝,微球疫苗粒徑為37.5±0.478μm,跨度0.46,微球包封率高達98.6%。微球在體外模擬胃液和腸液12h釋放率分別為10.2%和9.3%,24h釋放率分別為16.8%和13.2%。FITC標記的熒光微球示蹤表明微球能通過消化道到達后腸,1-24h在后腸能夠檢測到熒光微球,并可以明顯看到后腸對熒光微球的吸收。免疫組織化學(xué)結(jié)果顯示后腸在灌胃1天后免疫組化能檢測到棕黃色的陽性顆粒,只到第7天還能在后腸檢測到。微球疫苗口服免疫紅笛鯛后,用間接ELISA法測定血清、腸粘液和體表粘液抗體效價,結(jié)果表明,紅笛鯛在免疫后都發(fā)生特異免疫反應(yīng),灌胃組和投喂組在第四周時血清抗體效價都達到峰值1:4096和1:2048;腸粘液抗體效價在第3周和第5周分別達到峰值1:1024和1:512;體表粘液抗體效價和對照組沒有明顯變化。免疫30天后用活菌攻毒,測定受免魚的免疫保護率,口服微粒灌胃組和投喂組30天后的免疫保護率分別為63.1%和47.3%。綜合以上結(jié)果表明海藻酸鈉能作為弧菌口服疫苗的投遞載體,微球疫苗具有較好的免疫效果。
[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 鹵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.
【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：2463948
[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 鹵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.
【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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