嗜水气单胞菌微胶囊口服灭活疫苗制备工艺的筛选及其对小鼠的免疫
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摘要
1在已获得的嗜水气单胞菌灭活疫苗Ⅰ类新兽药证书的基础上将嗜水气单胞菌灭活疫苗进行微胶囊囊化，将其制备成嗜水气单胞菌微胶囊口服灭活疫苗。以嗜水气单胞菌(Ah)J-1株为芯材及大豆蛋白、麦芽糊精和阿拉伯胶等可被生物降解的物质为壁材，利用喷雾干燥微胶囊技术制备Ah微胶囊口服灭活疫苗。结果表明，喷雾干燥条件对疫苗微胶囊颗粒影响较大；其适宜条件为：进料温度40℃，进料速度60mL／h，大豆蛋白、麦芽糊精和阿拉伯胶之间最佳比例为3／9／4，进风温度和出风温度分别为195℃和90℃；制备的微胶囊疫苗颗粒的平均粒径为4.5-16.5um，平均含菌量为2.47×10~(13)mg~(-1)干粉，并将其拌入饵料中制成了可以直接通过饲喂而达到免疫效果的口服灭活疫苗。
2受免动物血清抗体效价是评价特异性免疫效果的一个重要指标，抗体效价的高低通常显示了免疫的成败，因此建立评价抗体水平的方法至关重要。按照嗜水气单胞菌灭活疫苗制备规程制备灭活疫苗，采用腹腔注射法免疫小鼠，第一次免疫2周后进行第二次免疫。取二次免疫后28d的小鼠血清2倍比稀释包被96孔酶标板，建立检测抗体的间接ELISA方法，测定受免小鼠特异性免疫应答水平。用传统的全菌凝集实验检测随机选取的30份免疫组血清和20份对照组血清，结果表明免疫组与对照组的血清抗体稀释最高滴度分别为1：32和1：8。用此阳、阴性血清建立间接ELISA方法，然后用该方法检测上面的50份血清。免疫组与对照组的血清抗体稀释最高滴度分别为1：4096和1：32。以上结果表明，建立的间接ELISA方法可更准确地用于微囊化口服灭活疫苗免疫小鼠的血清抗体效价检测。
3以嗜水气单胞菌(Ah)J-1株为材料，分别按照嗜水气单胞菌灭活疫苗制备规程制备灭活疫苗和喷雾干燥法制备微胶囊口服灭活疫苗。160只6-8周龄雌性小鼠分为4组，分别依次为注射AhJ-1全菌灭活疫苗组、口服微胶囊口服灭活疫苗组Ⅰ、Ⅱ和空白对照组，接种小鼠数每组40只。微胶囊口服灭活疫苗及全菌细胞灭活疫苗分别以直接拌入饵料法口服和腹腔注射免疫小鼠，空白对照组注射生理盐水(NS)，检测各组小鼠体液免疫水平。结果显示，用间接ELISA检测抗体水平，显示微胶囊口服灭活疫苗组Ⅰ(0.1g／只)的抗体水平高于对照组，略低于全菌灭活疫苗免疫组，但两者差异不显著(p＞0.05)；微胶囊口服灭活疫苗组Ⅱ(0.06g／只)的抗体水平高于对照组，低于全菌灭活疫苗免疫组，两者差异显著(p＜0.05)，但微胶囊口服灭活疫苗组Ⅰ和Ⅱ的抗体高效价维持时间最长即第6周。在第2次免疫后的第4周，用50LD_(50)AhJ-1攻击，AhJ-1攻击各组的保护率依次为，全菌灭活疫苗腹腔注射组的免疫保护力最高(73.3％)，其次为微胶囊疫苗组拌入饵料直接投喂组Ⅰ(0.1g／只)免疫保护力(63.3％)，与全菌灭活疫苗组(73.3％)差异不显著(p＞0.05)；微胶囊疫苗组拌入饵料直接投喂组Ⅱ(0.06g／只)免疫保护力(40％)，与全菌灭活疫苗组(73.3％)差异显著(p＜0.05)；空白对照组免疫保护力(0％)。表明所制备的嗜水气单胞菌微胶囊口服疫苗具有较好的癌苗性能。
Microencapsulated oral inactivated vaccines of Aeromonas hydrophila(Ah) were prepared by spray drying liquid o:w-emulsions in a laboratory spray dryer on the basis of one category new veterinary drug certificate of Aeromonas hydrophila awarded by china institute of veterinary drug control. Microencapsulated oral inactivated vaccines(MOIV) were prepared by a spray-drying method using a wall system consisting of gelatin、malt dextrin and soybean protein. The effects of technological parameters including the ratio of gelatin、malt dextrin and soybean protein, homogenization pressure,inlet temperature, feed temperature,and encapsulation efficiency (EE) were investigated.The resulting microcapsules were characterized in terms of AhJ-1 isomerization, storage stability and outer structures. The results showed that EE were significantly affected by the ratio of wall materials, homogenization pressure, inlet temperature, feed temperature and feed speed. The optimal condition was determined as follows.the ratio of soybean protein/malt dextrin/gelatin of 3/9/4,feed temperature of 40℃,inlet temperature of 195℃,outlet temperature of 90℃,homogenization pressure of 16 000r/pm and feed speed of 60ml/h. Scanning electron microscope(SEM) and acridine orange direct count (AODC) analysis showed that AhJ-1 microcapsules had a regular spherical shape with a mean diameter of 4.5-16.5um and smooth outer surface,the average bacterial mass of 2.47×10~(13) mg-lparticle-powder.The dry microencapsules remained physically stable for at least 6 months.The objective of the present study was to develop stable and usable fish oral vaccine microencapsulation to prevent the outbreak of Aeromonas hydrophila.。This paper describes a indirect enzyme-linked immunosorbent assay (ELISA) for rapid, specific and reliable assessment of the antibody immune response of vaccinated-with mice.Inactivated whole bacteria cells of Aeromonas hydrophila was prepared by the preparation rule of inactivated vaccine of Aeromonas hydrophila and intraperitoneally with mice 2 weeks again after the primary immunization. Before immunization, 2、6and 4 weeks after immunization, serum samples were collected from the caudal veins of mice. 30 shares of immunized-with group sera and 20 shares of control sera were randomly selected and tested with whole bacteria cells of Aeromonas hydrophila (Ah) antigen-based agglutination test. The results showed 30 immunized-with group sera with the highest dilution rate of 1:32 and 20 control one with the highest dilution rate of 1:8 was observed.To develop indirect-ELISA method,the above positive and negative sera which were determined by agglutination test were used. Further the same sera were tested with indirect-ELISA developed in this study.The results showed that 30 immunized-with group sera with the highest dilution rate of 1:4 096 and 20 control one with the highest dilution rate of 1:32 was detected. According to the results, it could be concluded that the indirect-ELISA developed in this study was used as a reliable diagnostic tool to anlayze antibdy titers of immunized-with mice.
To investigate the immunological effects induced by the microencapsulated oral inactivated vaccine, 160 elderly female mice were divided into randomly four groups,immunized-with orally group, intraperitoneally vaccinated-with group and control group, 40 mice in each group. Mice were immunized with microencapsulated oral inactivated vaccine for 7 days, inactivated whole cell vaccine of Aeronomas hydrophila and normal saline (NS) intraperitoneally respectively. The level of antibody titers in mice serum was determined by indirect ELISA . The results showed that the specific antibody response of immunized-with orally group mice was induced during the second week after immunization and effective protection against Aeomonas hydrophila, with responses similar to those observed in intraperitoneally-vaccinated with whole bacterial cells vaccine mice and an effective protection against Aeomonas hydrophila, with responses similar to those observed in intraperitoneally-vaccinated with whole bacterial cells vaccine mice. There was a significant difference in the antibody levels between the mice immunized with the microencapsulated oral vaccine and whole bacterial cells vaccine and the control group.The protection afforded by the microencapsulated oral inactivated vaccine and the existing whole bacterial cells vaccine was superior to that provided by intraperitoneally-vaccinated with NS. A time-course study indicated persistence of high specific antibody titers up to 6 weeks post-vaccination when microsperes were fed to mice. Challenge test were processed after 6 weeks post-vaccination and the relative percentage survival (RPS) was analyzed. The relative percentage survival of the vaccine fed groups were 63.3% (microencapsulated oral inactivated vaccine groupⅠ)、40%(microencapsulated oral inactivated vaccine groupⅡ)、73.3%(whole bacterial cells group)and 0%( control group) respectively.
2受免动物血清抗体效价是评价特异性免疫效果的一个重要指标，抗体效价的高低通常显示了免疫的成败，因此建立评价抗体水平的方法至关重要。按照嗜水气单胞菌灭活疫苗制备规程制备灭活疫苗，采用腹腔注射法免疫小鼠，第一次免疫2周后进行第二次免疫。取二次免疫后28d的小鼠血清2倍比稀释包被96孔酶标板，建立检测抗体的间接ELISA方法，测定受免小鼠特异性免疫应答水平。用传统的全菌凝集实验检测随机选取的30份免疫组血清和20份对照组血清，结果表明免疫组与对照组的血清抗体稀释最高滴度分别为1：32和1：8。用此阳、阴性血清建立间接ELISA方法，然后用该方法检测上面的50份血清。免疫组与对照组的血清抗体稀释最高滴度分别为1：4096和1：32。以上结果表明，建立的间接ELISA方法可更准确地用于微囊化口服灭活疫苗免疫小鼠的血清抗体效价检测。
3以嗜水气单胞菌(Ah)J-1株为材料，分别按照嗜水气单胞菌灭活疫苗制备规程制备灭活疫苗和喷雾干燥法制备微胶囊口服灭活疫苗。160只6-8周龄雌性小鼠分为4组，分别依次为注射AhJ-1全菌灭活疫苗组、口服微胶囊口服灭活疫苗组Ⅰ、Ⅱ和空白对照组，接种小鼠数每组40只。微胶囊口服灭活疫苗及全菌细胞灭活疫苗分别以直接拌入饵料法口服和腹腔注射免疫小鼠，空白对照组注射生理盐水(NS)，检测各组小鼠体液免疫水平。结果显示，用间接ELISA检测抗体水平，显示微胶囊口服灭活疫苗组Ⅰ(0.1g／只)的抗体水平高于对照组，略低于全菌灭活疫苗免疫组，但两者差异不显著(p＞0.05)；微胶囊口服灭活疫苗组Ⅱ(0.06g／只)的抗体水平高于对照组，低于全菌灭活疫苗免疫组，两者差异显著(p＜0.05)，但微胶囊口服灭活疫苗组Ⅰ和Ⅱ的抗体高效价维持时间最长即第6周。在第2次免疫后的第4周，用50LD_(50)AhJ-1攻击，AhJ-1攻击各组的保护率依次为，全菌灭活疫苗腹腔注射组的免疫保护力最高(73.3％)，其次为微胶囊疫苗组拌入饵料直接投喂组Ⅰ(0.1g／只)免疫保护力(63.3％)，与全菌灭活疫苗组(73.3％)差异不显著(p＞0.05)；微胶囊疫苗组拌入饵料直接投喂组Ⅱ(0.06g／只)免疫保护力(40％)，与全菌灭活疫苗组(73.3％)差异显著(p＜0.05)；空白对照组免疫保护力(0％)。表明所制备的嗜水气单胞菌微胶囊口服疫苗具有较好的癌苗性能。
Microencapsulated oral inactivated vaccines of Aeromonas hydrophila(Ah) were prepared by spray drying liquid o:w-emulsions in a laboratory spray dryer on the basis of one category new veterinary drug certificate of Aeromonas hydrophila awarded by china institute of veterinary drug control. Microencapsulated oral inactivated vaccines(MOIV) were prepared by a spray-drying method using a wall system consisting of gelatin、malt dextrin and soybean protein. The effects of technological parameters including the ratio of gelatin、malt dextrin and soybean protein, homogenization pressure,inlet temperature, feed temperature,and encapsulation efficiency (EE) were investigated.The resulting microcapsules were characterized in terms of AhJ-1 isomerization, storage stability and outer structures. The results showed that EE were significantly affected by the ratio of wall materials, homogenization pressure, inlet temperature, feed temperature and feed speed. The optimal condition was determined as follows.the ratio of soybean protein/malt dextrin/gelatin of 3/9/4,feed temperature of 40℃,inlet temperature of 195℃,outlet temperature of 90℃,homogenization pressure of 16 000r/pm and feed speed of 60ml/h. Scanning electron microscope(SEM) and acridine orange direct count (AODC) analysis showed that AhJ-1 microcapsules had a regular spherical shape with a mean diameter of 4.5-16.5um and smooth outer surface,the average bacterial mass of 2.47×10~(13) mg-lparticle-powder.The dry microencapsules remained physically stable for at least 6 months.The objective of the present study was to develop stable and usable fish oral vaccine microencapsulation to prevent the outbreak of Aeromonas hydrophila.。This paper describes a indirect enzyme-linked immunosorbent assay (ELISA) for rapid, specific and reliable assessment of the antibody immune response of vaccinated-with mice.Inactivated whole bacteria cells of Aeromonas hydrophila was prepared by the preparation rule of inactivated vaccine of Aeromonas hydrophila and intraperitoneally with mice 2 weeks again after the primary immunization. Before immunization, 2、6and 4 weeks after immunization, serum samples were collected from the caudal veins of mice. 30 shares of immunized-with group sera and 20 shares of control sera were randomly selected and tested with whole bacteria cells of Aeromonas hydrophila (Ah) antigen-based agglutination test. The results showed 30 immunized-with group sera with the highest dilution rate of 1:32 and 20 control one with the highest dilution rate of 1:8 was observed.To develop indirect-ELISA method,the above positive and negative sera which were determined by agglutination test were used. Further the same sera were tested with indirect-ELISA developed in this study.The results showed that 30 immunized-with group sera with the highest dilution rate of 1:4 096 and 20 control one with the highest dilution rate of 1:32 was detected. According to the results, it could be concluded that the indirect-ELISA developed in this study was used as a reliable diagnostic tool to anlayze antibdy titers of immunized-with mice.
To investigate the immunological effects induced by the microencapsulated oral inactivated vaccine, 160 elderly female mice were divided into randomly four groups,immunized-with orally group, intraperitoneally vaccinated-with group and control group, 40 mice in each group. Mice were immunized with microencapsulated oral inactivated vaccine for 7 days, inactivated whole cell vaccine of Aeronomas hydrophila and normal saline (NS) intraperitoneally respectively. The level of antibody titers in mice serum was determined by indirect ELISA . The results showed that the specific antibody response of immunized-with orally group mice was induced during the second week after immunization and effective protection against Aeomonas hydrophila, with responses similar to those observed in intraperitoneally-vaccinated with whole bacterial cells vaccine mice and an effective protection against Aeomonas hydrophila, with responses similar to those observed in intraperitoneally-vaccinated with whole bacterial cells vaccine mice. There was a significant difference in the antibody levels between the mice immunized with the microencapsulated oral vaccine and whole bacterial cells vaccine and the control group.The protection afforded by the microencapsulated oral inactivated vaccine and the existing whole bacterial cells vaccine was superior to that provided by intraperitoneally-vaccinated with NS. A time-course study indicated persistence of high specific antibody titers up to 6 weeks post-vaccination when microsperes were fed to mice. Challenge test were processed after 6 weeks post-vaccination and the relative percentage survival (RPS) was analyzed. The relative percentage survival of the vaccine fed groups were 63.3% (microencapsulated oral inactivated vaccine groupⅠ)、40%(microencapsulated oral inactivated vaccine groupⅡ)、73.3%(whole bacterial cells group)and 0%( control group) respectively.
引文
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