斑点叉尾鮰源嗜麦芽寡养单胞菌的PLGA微球和脂质体疫苗的制备及免疫原性研究
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摘要
本文研究了斑点叉尾鮰源嗜麦芽寡养单胞菌的PLGA微球疫苗和脂质体疫苗对斑点叉尾鮰免疫机能和免疫保护效应的影响。主要结果如下:
1.本试验采用复乳挥发法制备了嗜麦芽寡养单胞菌的PLGA微球疫苗,并测定其粒径大小约为(2.2±0.5)μm。600尾体重100±5g的斑点叉尾鮰分为15组,注射、口服、浸泡三种免疫途径各5组,每个免疫途径又包括低、中、高三种不同免疫剂量的PLGA微球疫苗组(Ⅰ组-Ⅲ组)、生理盐水对照组(Ⅳ组)和未包被嗜麦芽寡养单胞菌灭活苗组(Ⅴ组),疫苗免疫每两周1次共计3次。定期采血后测定血液免疫指标的变化,并用细菌攻毒试验检测免疫保护效应。结果显示本实验制备的PLGA微球疫苗包封率为88.93%,各免疫途径的疫苗免疫组均较生理盐水对照组能明显增强白细胞的吞噬活性、杀菌活性、血清溶菌酶、补体C_3、血清蛋白含量和抗体IgM水平。腹腔注射活菌攻毒后,注射免疫途径剂量为200μg/尾和100μg/尾受免组鱼(73.33%,66.68%)相对成活率较嗜麦芽寡养单胞菌灭活苗(64.17%)受免组鱼分别高出9.16%、2.51%,而50μg/尾的受免组鱼(49.17%)相比灭活苗组则低15%;口服免疫途径PLGA微球疫苗免疫组(Ⅰ组-Ⅲ组)的相对存活率分别为43.33%、65%和75%,而灭活苗口服组Ⅴ组仅有6.68%,表明PLGA微球疫苗对嗜麦芽寡养单胞菌病具有显著的免疫保护效应;而浸泡各免疫组的保护效应则不明显。
2.试验还采用反相蒸发法制备了脂质体疫苗,并用透射电镜测定其粒径大小约为(1±0.3)μm。将体重600±5g的斑点叉尾鮰分为15组,采用口服、注射和浸泡三种免疫方式各5组,每个免疫方式又包括低、中、高三种剂量的脂质体疫苗组(1组-3组)、生理盐水对照组(4组)和灭活苗组(5组),免疫每两周1次共3次。定期采血测定血液免疫指标的变化,并用细菌攻毒检测免疫保护效应。结果显示本实验制备的脂质体疫苗包封率为53.23%,各免疫途径的疫苗免疫组均能较对照组明显提高受免组鱼的血液免疫指标水平。腹腔注射活菌攻毒后,注射免疫剂量为200μg/尾、100μg/尾的受免组鱼相对成活率分别为80%、70.83%,较灭活苗受免组鱼分别高15.83%和6.66%,而50μg/尾的受免组鱼(53.33%)相比灭活苗组则低10.84%;口服途径脂质体疫苗免疫组(1组-3组)的相对存活率分别为39.18%、51.68%和58.33%,而灭活苗口服组5组仅有6.68%,表明脂质体疫苗能有效的保护抗原的免疫原性避免被破坏;浸泡免疫时可能受到水中阳离子和有机物的影响而导致免疫保护效应不理想。
The effect of the liposome-encapsulated antigen of Stenotrophomonas maltophilia and PLGA microspheres-encapsulated antigen of Stenotrophomonas maltophilia on immune function and potential protective efficacy against S.maltophilia infection in Channel Catfish(Ictalurus punctatus) were studied respectively.The results wvere as follows:
1.PLGA microspheres-encapsulated vaccine was prepared by using a water-in oil-in water(w/o/w) emulsification/solvent evaporation method.Channel Catfish was divided into fifteen groups.Each method which was employed for administration of antigen:immersion, injection and oral vaccination had five groups,different dosage of PLGA microspheres-encapsulated vaccine(groupⅠ-groupⅢ),saline(groupⅣ) and S.maltophilia antigen alone(groupⅤ),3 times at 2-week intervals.On weeks 0-7, phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method;lysozyme,C_3,protein and IgM in serum were detected by using kit.On week 9,all fish were challenged by injection with 1×10~9cfu/ml S.maltophilia to investigate the protective efficacy.The vaccines of PLGA microspheres-encapsulated bacterial antigen were obtained successfully.The mean diameter was 2.2±0.5μm.After immunization,phagocytic activity,bactericidal activity,lysozyme,C_3,protein and IgM were detected significantly.Channel Catfish immunized by injection were challenged with S.maltophilia alive,the relative survival rates of group 3(73.33%) was 9.16%higher than group 5(64.17%),group 2(66.68%) was 2.51%;however group 1(49.17%) was 15% lower than group 5.Another five groups which immunized by oral vaccination were challenged with S.maltophilia alive,the relative survival rates of group 1 to group 3 were 43.33%,65%,75%,but group 5 was a mere 6.68%,and the results suggest that PLGA microspheres-encapsulated antigen of S.maltophilia have the potential for induction of protective immune response against S.maltophilia infection.However the protective efficacy of Channel Catfish immunized by immersion was not good,due to it's mass.
2.Liposome-encapsulated vaccine was prepared by using reverse-phase evaporation method and the size of liposome was measured by electron microscopy.Channel Catfish was divided into fifteen groups.Each immune method had five groups,different dosage of liposome-encapsulated vaccine(group 1-group 3),saline(group 4) and S.maltophilia antigen alone(group 5),3 times at 2-week intervals.On weeks 0-7,phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method;lysozyme,C_3,protein and IgM were detected by using kit.On week 9,all fish were challenged by injection with S.maltophilia alive to investigate the protective efficacy.The vaccines of liposome-encapsulated bacterial antigen were obtained successfully.The size of liposome was round,and its mean diameter was 0.9±0.3μm.After immunization, phagocytic activity,bactericidal activity,lysozyme,C_3,protein and IgM were detected significantly.Charmel Catfish immunized by injection were challenged with S.maltophilia alive,the relative survival rates of group 2(70.83%) was 6.66%higher than group 5 (64.17%),group 3(80%) was 15.83%,however group 1(53.33%) was 10.84%lower than group 5.Another five groups which immunized by oral vaccination were challenged with S.maltophilia alive,the relative survival rates of group 1 to group 3 were 39.18%,51.68%, 58.33%,but group 5 was a mere 6.68%,and the results suggest that liposome-encapsulated vaccine could protect immunogeneity of antigen obviously.However the protective efficacy of Channel Catfish immunized by immersion was not good,because of positive ion and organics in water.
1.本试验采用复乳挥发法制备了嗜麦芽寡养单胞菌的PLGA微球疫苗,并测定其粒径大小约为(2.2±0.5)μm。600尾体重100±5g的斑点叉尾鮰分为15组,注射、口服、浸泡三种免疫途径各5组,每个免疫途径又包括低、中、高三种不同免疫剂量的PLGA微球疫苗组(Ⅰ组-Ⅲ组)、生理盐水对照组(Ⅳ组)和未包被嗜麦芽寡养单胞菌灭活苗组(Ⅴ组),疫苗免疫每两周1次共计3次。定期采血后测定血液免疫指标的变化,并用细菌攻毒试验检测免疫保护效应。结果显示本实验制备的PLGA微球疫苗包封率为88.93%,各免疫途径的疫苗免疫组均较生理盐水对照组能明显增强白细胞的吞噬活性、杀菌活性、血清溶菌酶、补体C_3、血清蛋白含量和抗体IgM水平。腹腔注射活菌攻毒后,注射免疫途径剂量为200μg/尾和100μg/尾受免组鱼(73.33%,66.68%)相对成活率较嗜麦芽寡养单胞菌灭活苗(64.17%)受免组鱼分别高出9.16%、2.51%,而50μg/尾的受免组鱼(49.17%)相比灭活苗组则低15%;口服免疫途径PLGA微球疫苗免疫组(Ⅰ组-Ⅲ组)的相对存活率分别为43.33%、65%和75%,而灭活苗口服组Ⅴ组仅有6.68%,表明PLGA微球疫苗对嗜麦芽寡养单胞菌病具有显著的免疫保护效应;而浸泡各免疫组的保护效应则不明显。
2.试验还采用反相蒸发法制备了脂质体疫苗,并用透射电镜测定其粒径大小约为(1±0.3)μm。将体重600±5g的斑点叉尾鮰分为15组,采用口服、注射和浸泡三种免疫方式各5组,每个免疫方式又包括低、中、高三种剂量的脂质体疫苗组(1组-3组)、生理盐水对照组(4组)和灭活苗组(5组),免疫每两周1次共3次。定期采血测定血液免疫指标的变化,并用细菌攻毒检测免疫保护效应。结果显示本实验制备的脂质体疫苗包封率为53.23%,各免疫途径的疫苗免疫组均能较对照组明显提高受免组鱼的血液免疫指标水平。腹腔注射活菌攻毒后,注射免疫剂量为200μg/尾、100μg/尾的受免组鱼相对成活率分别为80%、70.83%,较灭活苗受免组鱼分别高15.83%和6.66%,而50μg/尾的受免组鱼(53.33%)相比灭活苗组则低10.84%;口服途径脂质体疫苗免疫组(1组-3组)的相对存活率分别为39.18%、51.68%和58.33%,而灭活苗口服组5组仅有6.68%,表明脂质体疫苗能有效的保护抗原的免疫原性避免被破坏;浸泡免疫时可能受到水中阳离子和有机物的影响而导致免疫保护效应不理想。
The effect of the liposome-encapsulated antigen of Stenotrophomonas maltophilia and PLGA microspheres-encapsulated antigen of Stenotrophomonas maltophilia on immune function and potential protective efficacy against S.maltophilia infection in Channel Catfish(Ictalurus punctatus) were studied respectively.The results wvere as follows:
1.PLGA microspheres-encapsulated vaccine was prepared by using a water-in oil-in water(w/o/w) emulsification/solvent evaporation method.Channel Catfish was divided into fifteen groups.Each method which was employed for administration of antigen:immersion, injection and oral vaccination had five groups,different dosage of PLGA microspheres-encapsulated vaccine(groupⅠ-groupⅢ),saline(groupⅣ) and S.maltophilia antigen alone(groupⅤ),3 times at 2-week intervals.On weeks 0-7, phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method;lysozyme,C_3,protein and IgM in serum were detected by using kit.On week 9,all fish were challenged by injection with 1×10~9cfu/ml S.maltophilia to investigate the protective efficacy.The vaccines of PLGA microspheres-encapsulated bacterial antigen were obtained successfully.The mean diameter was 2.2±0.5μm.After immunization,phagocytic activity,bactericidal activity,lysozyme,C_3,protein and IgM were detected significantly.Channel Catfish immunized by injection were challenged with S.maltophilia alive,the relative survival rates of group 3(73.33%) was 9.16%higher than group 5(64.17%),group 2(66.68%) was 2.51%;however group 1(49.17%) was 15% lower than group 5.Another five groups which immunized by oral vaccination were challenged with S.maltophilia alive,the relative survival rates of group 1 to group 3 were 43.33%,65%,75%,but group 5 was a mere 6.68%,and the results suggest that PLGA microspheres-encapsulated antigen of S.maltophilia have the potential for induction of protective immune response against S.maltophilia infection.However the protective efficacy of Channel Catfish immunized by immersion was not good,due to it's mass.
2.Liposome-encapsulated vaccine was prepared by using reverse-phase evaporation method and the size of liposome was measured by electron microscopy.Channel Catfish was divided into fifteen groups.Each immune method had five groups,different dosage of liposome-encapsulated vaccine(group 1-group 3),saline(group 4) and S.maltophilia antigen alone(group 5),3 times at 2-week intervals.On weeks 0-7,phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method;lysozyme,C_3,protein and IgM were detected by using kit.On week 9,all fish were challenged by injection with S.maltophilia alive to investigate the protective efficacy.The vaccines of liposome-encapsulated bacterial antigen were obtained successfully.The size of liposome was round,and its mean diameter was 0.9±0.3μm.After immunization, phagocytic activity,bactericidal activity,lysozyme,C_3,protein and IgM were detected significantly.Charmel Catfish immunized by injection were challenged with S.maltophilia alive,the relative survival rates of group 2(70.83%) was 6.66%higher than group 5 (64.17%),group 3(80%) was 15.83%,however group 1(53.33%) was 10.84%lower than group 5.Another five groups which immunized by oral vaccination were challenged with S.maltophilia alive,the relative survival rates of group 1 to group 3 were 39.18%,51.68%, 58.33%,but group 5 was a mere 6.68%,and the results suggest that liposome-encapsulated vaccine could protect immunogeneity of antigen obviously.However the protective efficacy of Channel Catfish immunized by immersion was not good,because of positive ion and organics in water.
引文
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