CN105999281B - 禽类活病毒用冻干保护剂、制备方法及应用 - Google Patents
禽类活病毒用冻干保护剂、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种禽类活病毒用冻干保护剂、制备方法及应用，属于兽药技术领域。每1000mL冻干保护剂由以下组分组成：蔗糖40～100g，精氨酸0.5～2g，甘露醇0.5～1g，余量为0.01mol/L磷酸盐缓冲溶液。本发明中禽类活病毒用冻干保护剂不含有明胶、人血白蛋白、右旋糖酐等大分子过敏原性成分，配方简单，安全性高，效果好。与禽类活病毒培养物混合均匀后冻干，于2～8℃下保存24个月后外观、真空度、水分含量、病毒滴度均无明显变化，能极大地提高兽用生物制品企业毒种的质量。并且，该冻干保护剂的制备工艺简单、操作简便，适于规模化工业生产及应用，生产成本较低。
Description
技术领域
本发明涉及一种禽类活病毒用冻干保护剂，同时还涉及该冻干保护剂的制备方法及应用，属于兽药技术领域。
背景技术
禽类活病毒培养物(毒种)是兽用生物制品生产企业制造兽用生物制品的基础，毒种质量的优劣直接关系到制品的质量，合格的毒种是生产高质量制品的前提和重要保证。兽用生物制品生产企业通过建立完善的毒种种子批管理制度，实现毒种种子组成的均一性、同一性和稳定性，以此来满足相当长时间内的生产需要。
基础种子由原始种子经适当方式传代扩增而来，增殖到一定数量后，将相同代次的所有培养物均匀混合成一批，定量分装，保存于超低温冰箱中或经冷冻干燥后置适宜条件下保存。通常情况下，应对基础种子批进行病毒含量测定及安全或毒力测试，同时进行免疫抑制、毒力返强及免疫原性试验等。种子批应达到足够的规模，并含有足量的活病毒，因此毒种必须保存于超低温冰箱中或经冷冻干燥后置适宜条件下保存。但是超低温冰箱的能耗较高，且保存毒种的稳定性差，保存期短。
冷冻干燥是目前用于微生物、蛋白质等生物制品保存的最常用、最有效的方法。冻干保护剂和冷冻干燥技术是保证活病毒培养物质量的关键。冻干保护剂包括有机高分子物质、抗氧化剂等组分，其中有机高分子物质能够保证病毒活力及抗原稳定性，遇热不会焦糖化且耐干燥，能够与冷冻的低分子物质形成冻干活病毒耐热性结构，而抗氧化剂具有高度抗氧化作用，能最大限度地消耗介质中的氧，减少病毒与氧的接触，降低病毒的代谢活力及能量消耗，防止其在冷冻干燥及贮存过程中死亡。在冷冻干燥过程中，冷冻和干燥不可避免地会造成部分微生物细胞的损伤及死亡，或者引起蛋白质变性。公布号CN102657870A的发明专利公开了一种疫苗用冻干保护剂，包含以下组分(g/L)：右旋糖酐(40)10～60，蔗糖10～60，乳糖10～50，甘露醇5～30，甘氨酸5～20，精氨酸1～10，谷氨酸钠1～10，尿素1～10，199综合培养基1～50，该冻干保护剂不含有明胶及人血蛋白成分，疫苗内毒素含量低，对人体的刺激性和危害性小，但配方复杂，且不适于禽类活病毒的冻干保存。
因此，为提高冷冻干燥后毒种的存活率、生物活性以及长时间保存的稳定性，研制一种配方简单、安全性高、效果好的禽类活病毒培养物用冻干保护剂势在必行。
发明内容
本发明的目的是提供一种配方简单、安全性高、效果好的禽类活病毒用冻干保护剂。
同时，本发明还提供一种上述冻干保护剂的制备方法及应用。
为了实现以上目的，本发明所采用的技术方案是：
禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖40～100g，精氨酸0.5～2g，甘露醇0.5～1g，余量为0.01mol/L磷酸盐缓冲溶液。
优选的，禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖100g，精氨酸0.5g，甘露醇1g，余量为0.01mol/L磷酸盐缓冲溶液。
所述0.01mol/L磷酸盐缓冲溶液的组成为：NaCl 8g，KCl 0.2g，Na2HPO4 1.44g，KH2PO40.24g，pH 7.2～7.6。也可采用市售商品。
禽类活病毒用冻干保护剂的制备方法，包括以下步骤：按照用量准确取蔗糖、精氨酸和甘露醇，与0.01mol/L磷酸盐缓冲溶液混匀后定容，灭菌，即可。混合时可适当加热，以促进组分溶解。
禽类活病毒用冻干保护剂的应用，具体为：将禽类活病毒培养物、冻干保护剂混匀后冻干，即可。
所述禽类活病毒培养物与冻干保护剂的体积比为1:1。
所述冻干的操作为：预冻后于-50～-70℃、真空条件下冻干，即可。
本发明的有益效果：
本发明中禽类活病毒用冻干保护剂由蔗糖、甘露醇、精氨酸和0.01mol/L磷酸盐缓冲溶液组成，配方简单，安全性高，效果好。其中，0.01mol/L磷酸盐缓冲溶液更接近于活体生物的体液，可通过H2PO4 -、HPO4 2-间的转变实现H+转移，从而发挥缓冲作用，并维持渗透压平衡，尽量保证与禽类活病毒培养物原始生境的相似性，而蒸馏水不具有盐平衡作用，会破坏生物蛋白结构并影响其活性，同时生理盐水不具有pH调节作用，不能保证活性物质在最适条件下参与生物反应；蔗糖对阻止蛋白质二级结构改变，以及冻干、贮存过程中蛋白质多肽链的延伸和聚集发挥重要作用；而甘露醇属于多羟基化合物，其羟基能够替代蛋白质表面水分子的羟基，与病毒蛋白质表面的氢键形成一层假定的水化膜，保护氢键的连接位点不直接暴露在周围环境中，从而稳定蛋白质的高级结构，防止蛋白质因冻干、贮存而变性；在冻干过程中，0.01mol/L磷酸盐缓冲溶液结晶会引起pH值变化而使蛋白质变性，精氨酸能够抑制磷酸盐缓冲溶液结晶所致pH值改变，从而阻止蛋白质变性。
本发明中禽类活病毒用冻干保护剂不含有明胶、人血白蛋白、右旋糖酐等大分子过敏原性成分，制备工艺简单、操作简便，适于规模化工业生产及应用，生产成本较低。与禽类活病毒培养物混合均匀后冻干，于2～8℃下保存24个月后外观、真空度、水分含量、病毒滴度均无明显变化，安全、稳定、有效，能极大地提高兽用生物制品企业毒种的质量。
具体实施方式
下述实施例仅对本发明作进一步详细说明，但不构成对本发明的任何限制。
实施例1
禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖40g，精氨酸2g，甘露醇0.5g，余量为0.01mol/L磷酸盐缓冲溶液。0.01mol/L磷酸盐缓冲溶液的组成为：NaCl 8g，KCl 0.2g，Na2HPO4 1.44g，KH2PO4 0.24g，pH 7.4。
上述冻干保护剂的制备步骤为：按照用量准确称取蔗糖40g、精氨酸2g和甘露醇0.5g，将蔗糖、精氨酸、甘露醇依次加入800mL 0.01mol/L磷酸盐缓冲溶液中，加热搅拌溶解后定容至1000mL，于121℃高压灭菌30min，存于4℃备用。
上述冻干保护剂的应用，具体为：无菌条件下，将禽流感-HP株活病毒培养物(10日龄鸡胚制备)与等量(体积比1:1)冻干保护剂混合均匀，定量分装于10mL无菌西林瓶中(2mL/瓶)；半压塞后无菌保存于-60℃超低温冰箱中，使样品快速冻结，再在无菌条件下快速置于已于-60℃预冷30min的真空冷冻干燥机中，-60℃下冷冻干燥18h；冷冻干燥完毕，压塞加盖，贴签保存于4℃冰箱中备用。
实施例2
禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖60g，精氨酸1.5g，甘露醇0.6g，余量为0.01mol/L磷酸盐缓冲溶液。0.01mol/L磷酸盐缓冲溶液的组成同实施例1。
上述冻干保护剂的制备及应用同实施例1。
实施例3
禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖80g，精氨酸1g，甘露醇0.8g，余量为0.01mol/L磷酸盐缓冲溶液。0.01mol/L磷酸盐缓冲溶液的组成同实施例1。
上述冻干保护剂的制备及应用同实施例1。
实施例4
禽类活病毒用冻干保护剂，每1000mL由以下组分组成：蔗糖100g，精氨酸0.5g，甘露醇1g，余量为0.01mol/L磷酸盐缓冲溶液。0.01mol/L磷酸盐缓冲溶液的组成同实施例1。
上述冻干保护剂的制备及应用同实施例1。
实施例5
禽类活病毒用冻干保护剂的组成及制备同实施例4。
上述冻干保护剂的应用，具体为：无菌条件下，将鸭肝炎病毒-SD株活病毒培养物(4日龄雏鸭制备)与等量(体积比1:1)冻干保护剂混合均匀，定量分装于10mL无菌西林瓶中(2mL/瓶)；半压塞后无菌保存于-60℃超低温冰箱中，使样品快速冻结，再在无菌条件下快速置于已于-60℃预冷30min的真空冷冻干燥机中，-60℃下冷冻干燥18h；冷冻干燥完毕，压塞加盖，贴签保存于4℃冰箱中备用。
实施例6
禽类活病毒用冻干保护剂的组成及制备同实施例4。
上述冻干保护剂的应用，具体为：无菌条件下，将小鹅瘟-WF株活病毒培养物(10日龄鹅胚制备)与等量(体积比1:1)冻干保护剂混合均匀，定量分装于10mL无菌西林瓶中(2mL/瓶)；半压塞后无菌保存于-60℃超低温冰箱中，使样品快速冻结，再在无菌条件下快速置于已于-60℃预冷30min的真空冷冻干燥机中，-60℃下冷冻干燥18h；冷冻干燥完毕，压塞加盖，贴签保存于4℃冰箱中备用。
对比例1
冻干保护剂，每升由以下用量的组分组成：右旋糖酐(40)50g，蔗糖50g，乳糖20g，甘露醇5g，甘氨酸5g，精氨酸1.85g，谷氨酸钠9.2g，尿素4.6g，199综合培养基2g，余量为注射用水。
上述冻干保护剂的制备步骤为：按照用量准确取各组分，混匀后定容；用1N盐酸调节pH值为7.4，除菌过滤后存于4℃备用。
上述冻干保护剂的应用，操作同实施例1。
对比例2
冻干保护剂及其制备同对比例1，应用同实施例5。
对比例3
冻干保护剂，每升由以下用量的组分组成：人血清白蛋白10g，蔗糖40g，海藻糖15g，右旋糖酐(70)50g，谷氨酸钠6g，尿素3g，精氨酸1g，甘露醇10g，余量为PBS缓冲液。
上述冻干保护剂的制备步骤为：按照用量准确取各组分，混匀后定容，除菌过滤，存于4℃备用。
上述冻干保护剂的应用，操作同实施例1。
对比例4
冻干保护剂及其制备同对比例3，应用同实施例5。
试验例
分别将实施例1～6及对比例1～4中冻干粉置于4℃条件下保存，并于保存的第6、12、18、24个月对其外观、真空度、剩余水分、病毒滴度进行观察和检测，方法参见《中国兽药典》(2010版)第三部附录部分。测试结果见下表1。
表1实施例1～6及对比例1～4中冻干粉在4℃下保存的稳定性试验结果
由表1可知，实施例1～4中冻干保护剂的组分用量虽发生变化，但冻干粉在4℃下保存24个月后其外观、真空度、水分含量、病毒滴度均无明显变化，滴度下降均小于0.5。尤其是实施例4中冻干粉在4℃下保存30个月后外观、真空度、水分含量、病毒滴度仍无明显变化。
与对比例1～4中冻干粉相比，实施例中冻干粉的保存效果与之相当，或稍好于对比例。但由于实施例中冻干保护剂的组分较少，配方简单，因此生产成本较低。
Claims (7)
1.禽类活病毒用冻干保护剂，其特征在于：每1000mL由以下组分组成：蔗糖40~100g，精氨酸0.5~2g，甘露醇0.5~1g，余量为0.01mol/L磷酸盐缓冲溶液；所述0.01mol/L磷酸盐缓冲溶液的组成为：NaCl 8g，KCl 0.2g，Na2HPO4 1.44g，KH2PO4 0.24g，pH 7.2~7.6。
2.根据权利要求1所述的冻干保护剂，其特征在于：每1000mL由以下组分组成：蔗糖100g，精氨酸0.5g，甘露醇1g，余量为0.01mol/L磷酸盐缓冲溶液。
3.如权利要求1~2中任一项所述冻干保护剂的制备方法，其特征在于：包括以下步骤：按照用量准确取蔗糖、精氨酸和甘露醇，与0.01mol/L磷酸盐缓冲溶液混匀后定容，灭菌，即得。
4.如权利要求1~2中任一项所述冻干保护剂的应用，其特征在于：将禽类活病毒培养物、冻干保护剂混匀后冻干，即可。
5.根据权利要求4所述的应用，其特征在于：所述禽类活病毒培养物与冻干保护剂的体积比为1:1。
6.根据权利要求4所述的应用，其特征在于：所述冻干的操作为：预冻后于-50~-70℃、真空条件下冻干，即可。
7.根据权利要求5所述的应用，其特征在于：所述冻干的操作为：预冻后于-50~-70℃、真空条件下冻干，即可。
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Denomination of invention: Freeze dried protective agent for live avian virus, preparation method and Application Effective date of registration: 20210420 Granted publication date: 20190219 Pledgee: Henan Xinzheng Rural Commercial Bank Co.,Ltd. Pledgor: HENAN HOUYI BIOENGINEERING Co.,Ltd. Registration number: Y2021980002826 |
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Denomination of invention: Lyophilized protective agent for live poultry virus, preparation method and Application Effective date of registration: 20220411 Granted publication date: 20190219 Pledgee: Henan Xinzheng Rural Commercial Bank Co.,Ltd. Pledgor: HENAN HOUYI BIOENGINEERING CO.,LTD. Registration number: Y2022980004085 |
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Denomination of invention: Freeze dried protective agents for live poultry viruses, preparation methods and applications Effective date of registration: 20230424 Granted publication date: 20190219 Pledgee: Henan Xinzheng Rural Commercial Bank Co.,Ltd. Pledgor: HENAN HOUYI BIOENGINEERING CO.,LTD. Registration number: Y2023980038887 |