CN111000998A - 一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法 - Google Patents
一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法 Download PDFInfo
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Abstract
本发明提供了一种用于鸡法氏囊病精制卵黄抗体泡腾片的制备方法，该制备方法利用鸡传染性法氏囊油乳剂灭活苗免疫健康高产蛋鸡，收集高免蛋，提取卵黄抗体；附以益生菌，添加冻干保护剂和泡腾材料制备而得。该方法创新性的设计了鸡法氏囊病卵黄抗体的新剂型，丰富了鸡法氏囊病防控新途径，本发明方法成本低，抗体效价高，使用方便，生物安全性高，有效保证了所得卵黄抗体的治疗效果。
Description
技术领域
本发明属于兽用生物制品技术领域，具体涉及一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法。
背景技术
家禽集约化养殖带来前所未有的高速发展的同时也造成了鸡传染性法氏囊病等高度接触性传染病的迅猛发生，造成该病病毒主要在法氏囊的淋巴细胞内增殖，并且对其它免疫器官造成不同程度的损伤，最终导致鸡的法氏囊萎缩而引起鸡的免疫抑制。该病无明显的季节性，一年四季均可发生，发病率可达90％以上，临床上可以导致体重减轻、生长缓慢和骨骼肌出血等症状，一旦发生疫病，则传染快，死亡率高，死亡率达80％以上，损失更大，给养鸡业带来了巨大的经济损失。
卵黄抗体充分利用了母源性免疫，具有性质稳定、特异性强、制备成本低、口服安全等优点在家禽、家畜传染病的防治中已得到广泛的应用黄芪多糖为增强免疫力的一种中药多糖，可以参与动物机体细胞的各种活动，具有清除自由基、提高抗氧化酶活性和抑制脂质过氧化的能力；蒲公英黄酮抗氧化和抑菌功效显著，益生菌则进一步加强了本发明的抑菌、调节肠道环境促消化作用，制成泡腾剂一方面可以改善拌料饲喂不均，还可以解决传统抗体冷链运输的麻烦。
发明内容
本发明的目的在于提供一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，该方法设计新颖，解决了现有技术中冷链运输抗的不便，易于运输和保存，无生物毒性，有利于环境卫生及食品安全。
本发明的另一个目的在于解决了现有技术中卵黄抗体注射应激大，效果差的弊端。
为达到上述目的，本发明是通过以下技术方案实现的：
1.一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于：该制备方法的步骤如下：
(1)取未经免疫的23周龄健康产蛋鸡，对其进行鸡传染性法氏囊油乳剂灭活苗3次免疫注射，免疫间隔为10天，用量1ml/只/次，检测血清抗体效价至1:256时收集高免蛋；
(2)清洗消毒步骤(1)中所得高免蛋，收集并搅拌得卵黄匀浆；
(3)对步骤(2)中所得卵黄匀浆中添加1.5倍体积的生理盐水和浓度为0.1％的正辛酸，边加边搅拌0.5～1.5小时，室温放置2～6小时后，以4200～5500r/min的转速离心30min，丢弃沉淀，在上清液中加入1.2％硫酸酯葡聚糖，搅拌10-20分钟，用孔径5μm和1μm的筒式滤芯过滤上清液，再经截留分子量为10kD的中空纤维超滤柱进行浓缩3～4倍后再用NaOH调整PH值为6.8～7.0，并以0.45μm和0.22μm筒式滤芯过滤除菌，得抗体效价1:128的滤液。
(4)制备益生菌液：取双歧杆菌和枯草芽孢杆菌分别进行培养，再将两种菌液混合，得到益生菌液；
(5)将步骤(3)所述滤液与步骤(4)得到的益生菌液在无菌条件下按体积比1:1混合均匀，再按体积比1:1添加冻干保护剂混合均匀，冷冻真空干燥处理，得到冻干粉，
(6)将步骤(5)得到的冻干粉与泡腾材料混合均匀，压片，分装，得到鸡传染性法氏囊病卵黄抗体复合泡腾片。
作为优选，步骤(2)中所述的消毒高免蛋和收集卵黄的方法为：取步骤1)所得高免蛋，用清水冲洗干净后浸入0.1％新洁尔灭水溶液中消毒15分钟，取出晾干，再用环氧乙烷气雾消毒30分钟后，送入蛋清蛋黄分离机分离得到卵黄，搅拌得到卵黄匀浆。
作为优选，步骤(4)中长双歧杆菌和枯草芽孢杆菌分别按照以下步骤进行培养：将MRS液体培养基预热至37℃，同时按1％接种量接种长双歧杆菌液，37℃静置培养18～20小时，收获菌液至2-8℃保存备用；将芽孢杆菌培养基预热至37℃，同时按2％接种量接种枯草芽孢杆菌种子液，37℃静置培养16～20小时，收获菌液至2-8℃保存备用。
作为优选，权利要求1所述的冻干保护剂为每1000ml磷酸缓冲液中含有中药多糖50～70g、中药黄酮30～40g、维生素组合7～9g、明胶4.5～5.5g和蔗糖50～60g。
作为优选，步骤(5)中所述的冻干保护剂为每1000ml磷酸缓冲液中含有10-30g黄芪多糖和5-20g蒲公英黄酮。
作为优选，步骤(6)中所述的泡腾材料的制备方法：
按重量份数分别称取柠檬酸22份、蔗糖15份、碳酸氢钠35份、羧甲基淀粉钠10份，甜橙粉末1份、10％PVP无水乙醇4份、PEG60001份，硬脂酸镁0.5份，将上述成分充分混合，40～65℃烘干后过6～10目筛，制得颗粒备用，加入L-亮氨酸，经Co-60辐射灭菌制得泡腾材料。
本发明的优点及有益效果在于：
1、本发明降低了产品的冷链运输和存储要求，增加了养殖户使用的便利性。
2、本发明饮水饲喂同时具有微生态制剂和鸡传染性法氏囊病抗体的功效，减少了人力物力，降低了对鸡群的应激。
3、本发明可增强鸡群免疫力，提高鸡群整体免疫效果，降低鸡群混合感染疾病的风险。
具体实施方式
本发明通过以下实施例进一步详述。需要说明的是：下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
本发明所涉及的鸡传染性法氏囊灭活疫苗，黄芪多糖，蒲公英黄酮，长双歧杆菌和枯草芽孢杆菌均可以在市场上购买得到。
实施例1
一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其制备方法的步骤为：
(1)取未经免疫的23周龄健康产蛋鸡，对其进行鸡传染性法氏囊油乳剂灭活苗3次免疫注射，免疫间隔为10天，用量1ml/只/次，检测血清抗体效价至1:256时收集高免蛋；
(2)取步骤(1)所得高免蛋，用清水冲洗干净后浸入0.1％新洁尔灭水溶液中消毒15分钟，取出晾干，再用环氧乙烷气雾消毒30分钟后，送入蛋清蛋黄分离机分离得到卵黄，搅拌得到卵黄匀浆；
(3)对步骤(2)中所得卵黄匀浆中添加1.5倍体积的生理盐水和浓度为0.1％的正辛酸，边加边搅拌0.5小时，室温放置2小时后，以4200r/min的转速离心30min，丢弃沉淀，在上清液中加入1.2％硫酸酯葡聚糖，搅拌10min，用孔径5μm和1μm的筒式滤芯过滤上清液，再经截留分子量为10kD的中空纤维超滤柱进行浓缩3倍后再用NaOH调整PH值为6.8，并以0.45μm和0.22μm筒式滤芯过滤除菌，得抗体效价1:128的滤液。
(4)制备益生菌液：将乳酸菌液体培养基预热至37℃，同时按1％接种量接种长双歧杆菌液，37℃静置培养18小时，收获菌液至2-8℃保存备用；将芽孢杆菌培养基预热至37℃，同时按2％接种量接种枯草芽孢杆菌种子液，37℃静置培养16小时，收获菌液至2-8℃保存备用。
(5)制备冻干保护剂：按每1000ml磷酸缓冲液中加入10g黄芪多糖和5g蒲公英黄酮来制得冻干保护剂，并将步骤(3)所述滤液与步骤(4)得到的益生菌液在无菌条件下按体积比1:1混合均匀，再按体积比1:1添加冻干保护剂混合均匀，冷冻真空干燥处理，得到冻干粉；
(6)制备泡腾材料：按重量份数分别称取柠檬酸22份、蔗糖15份、碳酸氢钠35份、羧甲基淀粉钠10份，甜橙粉末1份、10％PVP无水乙醇4份、PEG60001份，硬脂酸镁0.5份，将上述成分充分混合，40℃烘干后过6目筛，制得颗粒备用，加入L-亮氨酸，经Co-60辐射灭菌制得泡腾材料。
(7)将步骤(5)得到的冻干粉与泡腾材料混合均匀，压片，分装，得到鸡传染性法氏囊病卵黄抗体复合泡腾片。
实施例2
一种不含益生菌的鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其制备方法的步骤为：
(1)取未经免疫的23周龄健康产蛋鸡，对其进行鸡传染性法氏囊油乳剂灭活苗3次免疫注射，免疫间隔为10天，用量1ml/只/次，检测血清抗体效价至1:256时收集高免蛋；
(2)取步骤(1)所得高免蛋，用清水冲洗干净后浸入0.1％新洁尔灭水溶液中消毒15分钟，取出晾干，再用环氧乙烷气雾消毒30分钟后，送入蛋清蛋黄分离机分离得到卵黄，搅拌得到卵黄匀浆；
(3)对步骤(2)中所得卵黄匀浆中添加1.5倍体积的生理盐水和浓度为0.1％的正辛酸，边加边搅拌1.5小时，室温放置6小时后，以5500r/min的转速离心30min，丢弃沉淀，在上清液中加入1.2％硫酸酯葡聚糖，搅拌20min，用孔径5μm和1μm的筒式滤芯过滤上清液，再经截留分子量为10kD的中空纤维超滤柱进行浓缩3～4倍后再用NaOH调整PH值为7.0，并以0.45μm和0.22μm筒式滤芯过滤除菌，得抗体效价1:128的滤液。
(4)制备冻干保护剂：按每1000ml磷酸缓冲液中加入30g黄芪多糖和20g蒲公英黄酮来制得冻干保护剂，并将步骤(3)所述滤液按体积比1:1添加冻干保护剂混合均匀，冷冻真空干燥处理，得到冻干粉；
(5)制备泡腾材料：按重量份数分别称取柠檬酸22份、蔗糖15份、碳酸氢钠35份、羧甲基淀粉钠10份，甜橙粉末1份、10％PVP无水乙醇4份、PEG60001份，硬脂酸镁0.5份，将上述成分充分混合，65℃烘干后过10目筛，制得颗粒备用，加入L-亮氨酸，经Co-60辐射灭菌制得泡腾材料。
(6)将步骤(4)得到的冻干粉与泡腾材料混合均匀，压片，分装，得到一种不含益生菌的鸡法氏囊病精制卵黄抗体泡腾片。
实施例3
一种不含冻干保护剂的鸡传染性法氏囊病卵黄抗体泡腾片的制备方法，其制备方法的步骤为：
(1)取未经免疫的23周龄健康产蛋鸡，对其进行鸡传染性法氏囊油乳剂灭活苗3次免疫注射，免疫间隔为10天，用量1ml/只/次，检测血清抗体效价至1:256时收集高免蛋；
(2)取步骤(1)所得高免蛋，用清水冲洗干净后浸入0.1％新洁尔灭水溶液中消毒15分钟，取出晾干，再用环氧乙烷气雾消毒30分钟后，送入蛋清蛋黄分离机分离得到卵黄，搅拌得到卵黄匀浆；
(3)对步骤(2)中所得卵黄匀浆中添加1.5倍体积的生理盐水和浓度为0.1％的正辛酸，边加边搅拌1.5小时，室温放置6小时后，以5500r/min的转速离心30min，丢弃沉淀，在上清液中加入1.2％硫酸酯葡聚糖，搅拌20min，用孔径5μm和1μm的筒式滤芯过滤上清液，再经截留分子量为10kD的中空纤维超滤柱进行浓缩3～4倍后再用NaOH调整PH值为7.0，并以0.45μm和0.22μm筒式滤芯过滤除菌，得抗体效价1:128的滤液。
(4)制备益生菌液：将乳酸菌液体培养基预热至37℃，同时按1％接种量接种长双歧杆菌液，37℃静置培养20小时，收获菌液至2-8℃保存备用；将芽孢杆菌培养基预热至37℃，同时按2％接种量接种枯草芽孢杆菌种子液，37℃静置培养20小时，收获菌液至2-8℃保存备用。
(5)将步骤(3)所述滤液与步骤(4)得到的益生菌液在无菌条件下按体积比1:1混合均匀，冷冻真空干燥处理，得到干粉；
(6)制备泡腾材料：按重量份数分别称取柠檬酸22份、蔗糖15份、碳酸氢钠35份、羧甲基淀粉钠10份，甜橙粉末1份、10％PVP无水乙醇4份、PEG60001份，硬脂酸镁0.5份，将上述成分充分混合，65℃烘干后过10目筛，制得颗粒备用，加入L-亮氨酸，经Co-60辐射灭菌制得泡腾材料。
(7)将步骤(5)得到的干粉与泡腾材料混合均匀，压片，分装，得到一种不含冻干保护剂的鸡传染性法氏囊病卵黄抗体泡腾片。
临床试验
1、试验材料：
按实施例1制得本发明鸡传染性法氏囊病卵黄抗体复合泡腾片，用于1组；
按实施例2制得一种不含益生菌的鸡法氏囊病精制卵黄抗体泡腾片；用于2组；
按实施例3制得一种不含冻干保护剂的鸡法氏囊病精制卵黄抗体泡腾片；用于3组；
普通抗体和化药组均为市售产品，按说明书使用。
2、本发明的使用方法：1组：按本发明1片溶于1kg水中计算使用剂量，将本发明给禽自由饮用；2组和3组按1片溶于1kg水中计算使用剂量，将本发明给禽自由饮用。
3、试验分组及处理
2018年于山东烟台某发生鸡传染性法氏囊病鸡场选取3周龄患病AA肉鸡60羽，随机分为6组，每组10只，1-3组为实验组，4-5组为鸡传染性法氏囊病普通抗体治疗组和市售化药治疗组，6组为空白对照组，各组均同等饲养环境下饲喂全价料，观察15天，试验结果如下表。
表本发明治疗鸡传染性法氏囊病效果试验
结论
本发明鸡法氏囊病精制卵黄抗体泡腾片对鸡传染性法氏囊病的治愈率为100％，显著优于其他几组，与不含益生菌的鸡法氏囊病精制卵黄抗体泡腾片和不含冻干保护剂的鸡法氏囊病精制卵黄抗体泡腾片相比，采食量和体质恢复快，日增重20g/d，饲料利用率高；而不含益生菌的鸡法氏囊病精制卵黄抗体泡腾片和不含冻干保护剂的鸡法氏囊病精制卵黄抗体泡腾片与市售普通抗体组治愈率相比高出20％，可见鸡法氏囊病精制卵黄抗体泡腾片整体治疗效果明显，而益生菌和冻干保护剂的添加对提高免疫力和降低料肉比有较大的协同作用，这与益生菌协消化道菌群功能和黄芪多糖、蒲公英黄酮的协调免疫和促稳定的作用密切相关。
Claims (5)
1.一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于：该制备方法的步骤如下：
(1)取未经免疫的23周龄健康产蛋鸡，对其进行鸡传染性法氏囊油乳剂灭活苗3次免疫注射，免疫间隔为10天，用量1ml/只/次，检测血清抗体效价至1:256时收集高免蛋；
(2)清洗消毒步骤(1)中所得高免蛋，收集并搅拌得卵黄匀浆；
(3)对步骤(2)中所得卵黄匀浆中添加1.5倍体积的生理盐水和浓度为0.1％的正辛酸，边加边搅拌0.5～1.5小时，室温放置2～6小时后，以4200～5500r/min的转速离心30min，丢弃沉淀，在上清液中加入1.2％硫酸酯葡聚糖，搅拌10-20分钟，用孔径5μm和1μm的筒式滤芯过滤上清液，再经截留分子量为10kD的中空纤维超滤柱进行浓缩3～4倍后再用NaOH调整PH值为6.8～7.0，并以0.45μm和0.22μm筒式滤芯过滤除菌，得抗体效价1:128的滤液。
(4)制备益生菌液：取双歧杆菌和枯草芽孢杆菌分别进行培养，再将两种菌液混合，得到益生菌液；
(5)将步骤(3)所述滤液与步骤(4)得到的益生菌液在无菌条件下按体积比1:1混合均匀，再按体积比1:1添加冻干保护剂混合均匀，冷冻真空干燥处理，得到冻干粉；
(6)将步骤(5)得到的冻干粉与泡腾材料混合均匀，压片，分装，得到鸡传染性法氏囊病卵黄抗体复合泡腾片。
2.根据权利要求1所述的一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于步骤(2)中所述的消毒高免蛋和收集卵黄的方法为：取步骤1)所得高免蛋，用清水冲洗干净后浸入0.1％新洁尔灭水溶液中消毒15分钟，取出晾干，再用环氧乙烷气雾消毒30分钟后，送入蛋清蛋黄分离机分离得到卵黄，搅拌得到卵黄匀浆。
3.根据权利要求1所述的一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于所述的步骤(4)中长双歧杆菌和枯草芽孢杆菌分别按照以下步骤进行培养：将MRS液体培养基预热至37℃，同时按1％接种量接种长双歧杆菌液，37℃静置培养18～20小时，收获菌液至2-8℃保存备用；将芽孢杆菌培养基预热至37℃，同时按2％接种量接种枯草芽孢杆菌种子液，37℃静置培养16～20小时，收获菌液至2-8℃保存备用。
4.根据权利要求1所述的一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于步骤(5)中所述的冻干保护剂为每1000ml磷酸缓冲液中含有10-30g黄芪多糖和5-20g蒲公英黄酮。
5.根据权利要求1所述的一种鸡法氏囊病精制卵黄抗体泡腾片的制备方法，其特征在于步骤(6)中所述的泡腾材料的制备方法：
按重量份数分别称取柠檬酸22份、蔗糖15份、碳酸氢钠35份、羧甲基淀粉钠10份，甜橙粉末1份、10％PVP无水乙醇4份、PEG60001份，硬脂酸镁0.5份，将上述成分充分混合，40～65℃烘干后过6～10目筛，制得颗粒备用，加入L-亮氨酸，经Co-60辐射灭菌制得泡腾材料。
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