CN114042167B - 鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂及其制备方法和应用 - Google Patents
鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及动物用病毒活疫苗制造领域，特别公开了一种鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂及其制备方法和应用。该耐热保护剂，具体成分包括L‑谷氨酸钠1‑2%、葡萄糖6‑8%、聚乙烯吡咯烷酮（PVPK30）4‑6%、胰蛋白胨1‑2%、乳糖1‑2%、聚乙二醇（PEG）1‑2%、甘露醇1‑2%、纳米植物甾醇1‑2%、泊洛沙姆0.1‑0.3%、赖氨酸0.1‑0.3%、余量为注射用水。本发明的耐热冻干保护剂解决了鸡新城疫、传染性支气管炎二联活疫苗不能在2~8℃下长期保存和运输的问题。
Description
技术领域
本发明涉及动物用病毒活疫苗制造领域，特别涉及一种鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂及其制备方法和应用。
背景技术
冻干保护剂及其制备工艺是活疫苗生产中的一项关键技术，对疫苗的保存、运输和使用有很大影响。目前，在我国兽用疫苗中，活的冻干疫苗占相当大的比例，国内常用的保护剂主要为脱脂奶粉、蔗糖、明胶等，组方简单，对疫苗的保护功能差。如果在2-8℃条件下，保存期只有4-6个月，需要在-15℃以下低温保存。给生产厂家及使用者造成很大不便，加上国内传统的生物制品保护剂的研制相对滞后，使成品疫苗的长期保存和运输受限，所以，耐热冻干保护剂的研究是非常急需的，在20世纪80年代，发达国家就已经开始进行耐热冻干保护剂的研究，我国在耐热冻干保护剂方面的研究起步较晚，加上中国市场巨大，国外生物制品涌入，市场竞争加剧。为了我国生物制品也的生存和发展，新型有效的耐热冻干保护剂的研究和应用显得尤为迫切。
耐热冻干保护剂与常规的冻干保护剂相比，其冻干疫苗具有耐高温保存期长的特点，因而耐热保护剂的性能要比传统保护剂更为优良，使活疫苗可在有效期内保存在2-8℃条件下，即使在较高的室温下也可以保存几十天，因此使活疫苗的储存、运输和使用都更方便、经济。
近年我国也有一些耐热保护剂研究的报道，主要包括鸡新城疫、鸡马立克氏病、鸡传染性法氏囊病、鸡传染性支气管炎、鸡痘和猪瘟冻干活疫苗等的耐热冻干保护剂研究。其主要使用明胶、乳糖、各类氨基酸等作为保护剂，而且都是单苗。这些研究都使用明胶，明胶中含有很多异种蛋白，明胶注射到动物体内，容易造成动物的应激反应。本发明添加纳米植物甾醇能有效提升抗原的抗氧化性，降低抗原效价损失，泊洛沙姆添加提升疫苗的复融性。因此研制一种配制方法简单、成本低、不含明胶，能够大规模生产、具有良好保护性能的鸡新城疫、传染性支气管炎二联活疫苗耐热冻干保护剂久显得尤为迫切重要。
发明内容
本发明为了弥补现有技术的不足，提供了一种配制简单、生产成本低、适于规模化生产的鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂及其制备方法和应用。
本发明是通过如下技术方案实现的：
一种鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂，其特征在于：由A液和B液按照1:1的体积混合制成，
其中，A液由以下重量比的原料混合制成：胰蛋白胨2-4%、聚乙烯吡咯烷酮（PVPK30）8-10%，其余为水；
B液由以下重量比的原料混合制成：L-谷氨酸钠2-4%、葡萄糖12-16%、乳糖2-4%、聚乙二醇（PEG）2-4%、甘露醇2-4%、纳米植物甾醇2-4%、泊洛沙姆0.2-0.6%、赖氨酸0.2-0.6%，其余为水。
进一步优选的，所述A液和B液中的用水为注射用水。
上述鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂的制备方法，包括如下步骤：
（1）将胰蛋白胨、聚乙烯吡咯烷酮溶解在水中，经121℃、20min高压灭菌，制备得到A液；
（2）将L-谷氨酸钠、葡萄糖、乳糖、聚乙二醇、甘露醇、纳米植物甾醇、泊洛沙姆、赖氨酸溶解于水中，采用0.22μm滤膜过滤除菌，得到B液；
（3）将A液和B液按照1:1的体积比混合在一起，即得耐热保护剂产品。
上述鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂的应用，将耐热保护剂与鸡新城疫和鸡传染性支气管炎混合病毒液按照1:2的体积比混合，经分装后冷冻干燥。
进一步优选的，所述冷冻干燥时间不少于28小时。
本发明最大的优点是解决了现有的耐热保护剂中明胶的添加剂普遍存在的成分复杂和成本高的缺陷，具有耐热性好、能有效保护鸡新城疫、传染性支气管炎二联活疫苗，配方成分及配制方法简单，生产成本低，在任何生物制品厂都完全可以实现量产等优点。
本发明的保护剂保护的疫苗在2-8℃下保存24个月，仍保持冻干后的外形，病毒含量超过国家收益生物制品规程的标准，冻干前后病毒损失率低，冻干产品耐老化程度高，产品在2-8℃保存24个月，病毒含量不发生改变，从而解决了鸡新城疫、传染性支气管炎二联活疫苗不能再2-8℃下长期保存和运输的技术问题。
具体实施方式
下面结合具体实施例来进一步描述本发明，本反面的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1：耐热冻干保护剂的制备
A液的制备：胰蛋白胨1g和聚乙烯吡咯烷酮6g溶解在100ml注射水中，采用121℃，20分钟高压灭菌；
B液的配制：L-谷氨酸钠1g、葡萄糖8g、乳糖1g、聚乙二醇1.5g、甘露醇1g、纳米植物甾醇1g、泊洛沙姆0.3g和赖氨酸0.1g充分溶解在100ml注射水中，采用0.22μm滤膜过滤除菌；
将A液与B液按照1:1的体积比混合均匀，即得本发明耐热冻干保护剂。
实施例2：耐热冻干保护剂的制备
A液的制备：胰蛋白胨2g和聚乙烯吡咯烷酮5g溶解在100ml注射水中，采用121℃，20分钟高压灭菌；
B液的配制：L-谷氨酸钠1.5g、葡萄糖7g、乳糖1.5g、聚乙二醇1g、甘露醇1.5g、纳米植物甾醇1.5g、泊洛沙姆0.2g和赖氨酸0.2g充分溶解在100ml注射水中，采用0.22μm滤膜过滤除菌；
将A液与B液按照1:1的体积比混合均匀，即得本发明耐热冻干保护剂。
实施例3：耐热冻干保护剂的制备
A液的制备：胰蛋白胨2g和聚乙烯吡咯烷酮4g溶解在100ml注射水中，采用121℃，20分钟高压灭菌；
B液的配制：L-谷氨酸钠2g、葡萄糖6g、乳糖2g、聚乙二醇2g、甘露醇2g、纳米植物甾醇2g、泊洛沙姆0.1g和赖氨酸0.3g充分溶解在100ml注射水中，采用0.22μm滤膜过滤除菌；
将A液与B液按照1:1的体积比混合均匀，即得本发明耐热冻干保护剂。
试验例1：本发明耐热冻干保护剂对鸡新城疫、传染性支气管炎二联活疫苗的保护效力试验
1.试验材料
1.1毒株
鸡新城疫病毒（La Sota株），购自中国兽医药品监察所；
鸡传染性支气管炎病毒（H120株、H52株），购自中国兽医药品监察所。
1.2 SPF鸡胚，购自山东昊泰实验动物繁育有限公司。
1.3试剂，购自国药集团和Sigma公司。
2.试验疫苗的制备
试验疫苗分为A、B、C、D四个组别，其中各组试验疫苗的制备方法如下：
A、B、C组试验疫苗按照实施例1-3的三个配方的冻干保护剂分别与鸡新城疫、传染性支气管炎病毒液混合，无菌分装于西林瓶中，每组分别试验01、02、03三个批次。-40℃预冻3小时后，启动真空泵，达最大真空后启动加热装置，一次升华温度设定为-10℃，用时在17小时以内，二次干燥温度设定为25℃，用时在5小时左右。
D组试验疫苗：用常规5%蔗糖脱脂乳配制的保护剂（蔗糖5g，脱脂奶粉10g，加注射水至100ml）与鸡新城疫、传染性支气管炎病毒混合液混合后，无菌分装于西林瓶中，试验01、02、03三个批次。-40℃预冻3小时后，启动真空泵，达最大真空后启动加热装置，一次升华温度设定为-10℃，用时在17小时以内，二次干燥温度设定为25℃，用时在5小时左右。
3.试验组制品技术参数及检验方法
3.1产品的物理性状、无菌检验、支原体检验、外源病毒检验、特异性检验、安全检验、剩余水分测定和真空度测定按《中华人民共和国兽用生物制品规程》的方法及标准进行。
3.2病毒含量测定方法及标准：以鸡胚半数感染量（EID50）作为衡量疫苗效价的标准。效价测定方法按照《中华人民共和国兽用生物制品规程》鸡新城疫、传染性支气管炎二联活疫苗效价检测方法进行。
（1）鸡新城疫弱毒冻干疫苗的标准：
①冻干后每羽份病毒含量≥106.5EID50；
②冻干后制品放于37℃保存10天进行病毒含量测定，病毒含量下降在1个滴度以内；
③冻干后制品放于2-8℃保存24个月，每羽份病毒含量≥106.2EID50。
（2）鸡传染性支气管炎弱毒冻干疫苗的标准：
①冻干后每羽份病毒含量≥104.0EID50；
②冻干后制品放于37℃保存10天进行病毒含量测定，病毒含量下降在1个滴度以内；
③冻干后制品放于2~8℃保存24个月，每羽份病毒含量≥103.5EID50。
4.试验结果
表1 37℃保存效价测定（EID50/羽份）新城疫部分
表2 37℃保存效价测定（EID50/羽份）传染性支气管炎部分
表3 2-8℃保存效价测定（EID50/羽份）新城疫部分
表4 2~8℃保存效价测定（EID50/羽份）传染性支气管炎部分
从以上试验结果可以看出，在2-8℃温度条件下，本发明耐热冻干保护剂对鸡新城疫、传染性支气管炎二联活疫苗的保护效果，明显优于常规的5%蔗糖脱脂乳配制的保护剂。37℃放置10天后的效价结果可以看出本发明耐热冻干保护剂毒价损失小于1个滴度，明显优于常规的5%蔗糖脱脂乳配制的保护剂。
试验结果说明，采用本发明耐热保护剂所配制的鸡新城疫、传染性支气管炎二联活疫苗耐热性能良好，本发明耐热冻干保护剂对鸡新城疫、传染性支气管炎二联活疫苗具有良好的保护效果。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改，等同替换、改进等，均应包含在本发明的保护范围内。
Claims (4)
1.一种鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂，其特征在于：由A液和B液按照1:1的体积混合制成，其中，A液由以下重量比的原料混合制成：胰蛋白胨2-4%、聚乙烯吡咯烷酮8-10%，其余为注射用水；B液由以下重量比的原料混合制成：L-谷氨酸钠2-4%、葡萄糖12-16%、乳糖2-4%、聚乙二醇2-4%、甘露醇2-4%、纳米植物甾醇2-4%、泊洛沙姆0.2-0.6%、赖氨酸0.2-0.6%，其余为注射用水。
2.如权利要求1所述的鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂的制备方法，其特征为，包括如下步骤：（1）将胰蛋白胨、聚乙烯吡咯烷酮溶解在水中，经121℃、20min高压灭菌，制备得到A液；（2）将L-谷氨酸钠、葡萄糖、乳糖、聚乙二醇、甘露醇、纳米植物甾醇、泊洛沙姆、赖氨酸溶解于水中，采用0.22μm滤膜过滤除菌，得到B液；（3）将A液和B液按照1:1的体积比混合在一起，即得耐热保护剂产品。
3.如权利要求1所述的鸡新城疫、传染性支气管炎二联活疫苗耐热保护剂的应用，其特征在于：将耐热保护剂与鸡新城疫和鸡传染性支气管炎混合病毒液按照1:2的体积比混合，经分装后冷冻干燥。
4.如权利要求3所述的应用，其特征在于：所述冷冻干燥时间不少于28小时。
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