CN116036257A - 一种猪瘟活疫苗的制备方法 - Google Patents
一种猪瘟活疫苗的制备方法 Download PDFInfo
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Abstract
本发明公开了一种猪瘟活疫苗的制备方法,属于生物技术领域。上述制备方法包括:(1)细胞制备:用无血清培养基培养全悬浮细胞,经重悬、传代培养、扩大培养,获取细胞液;(2)病毒液制备:将猪瘟病毒接种细胞液,然后补加维持液,采用流加培养方式连续培养收获病毒液;(3)病毒纯化:所收获的病毒液中加入羧甲基纤维素钠,静置过夜,过滤,收集滤出液;(4)疫苗冻干:滤出液中加入冻干保护剂,定量分装,真空冷冻干燥,制成猪瘟活疫苗。本发明采用无血清全悬浮培养的技术方法增殖猪瘟病毒,生产工艺易于放大、生产效率高,制备的疫苗效价高、安全性好且免疫效果更佳。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种猪瘟活疫苗的制备方法。
背景技术
猪瘟(Classical swine fever,CSF)是由猪瘟病毒引起的一种高度接触传染性疾病。疫苗免疫是防控该病的首要手段。
目前产业化的猪瘟活疫苗制备方法主要有二种,一种是常规的细胞转瓶培养方法,该方法技术难度低,但生产效率相对较低、劳动强度高;另一种是微载体悬浮培养方法,该方法存在工艺放大困难、培养规模有限,杂蛋白含量相对较高。以上二种方法均应用含血清培养基的细胞贴壁培养工艺,病毒增殖滴度不高,制备的疫苗免疫效果不尽理想,而且异源血清增加了疫苗副反应发生的几率。
因此,亟需开发一种培养病毒滴度高、易于放大、产品安全性更好的全悬浮无血清培养方法,制备猪瘟活疫苗,来满足畜牧业防控猪瘟病毒病的技术需求。
发明内容
本发明的目的是提供一种猪瘟活疫苗的制备方法,以解决上述现有技术存在的问题,该猪瘟活疫苗生产工艺易于放大、生产效率高、生产成本低,制备的成品疫苗效价高、安全性好,疫苗的保存期长,免疫效果更佳。
为实现上述目的,本发明提供了如下方案:
本发明提供一种猪瘟活疫苗的制备方法,包括以下步骤:
(1)细胞制备
用无血清培养基培养全悬浮细胞,经重悬、传代培养、扩大培养,获取细胞液;
(2)病毒液制备
将猪瘟病毒接种所述细胞液,然后补加维持液,所述维持液包括无血清培养基和辅助液,接毒后继续培养72~96h,首次收获病毒液;之后采用流加培养方式继续培养,连续培养收获10~15次病毒液;
(3)病毒纯化
所述病毒液中加入羧甲基纤维素钠,静置过夜,过滤,收集滤出液;
(4)疫苗冻干
所述滤出液中加入冻干保护剂,定量分装,真空冷冻干燥,制成猪瘟活疫苗。
优选的,步骤(1)中,所述全悬浮细胞包括ST细胞、PK15细胞和MPK细胞;全悬浮细胞重悬及传代的细胞密度均为1.0×106个/mL。
优选的,步骤(2)中,按照MOI为0.01~0.1将所述猪瘟病毒接种所述细胞液。
优选的,步骤(2)中,所述无血清培养基与所述辅助液体积比为99:1;其中,所述辅助液为PEG2000 5~10g/L、硫酸锌0.1~0.3g/L和PVP K30 0.5~1g/L的水溶液。
优选的,步骤(2)中,所述流加培养方式具体为:每次收获所述病毒液时,反应器中留存0.1~1%的病毒液,先补加培养体积比为1/20~1/8的所述细胞液,然后补加所述维持液至培养体积,37℃继续培养48~72小时,再次收获。
优选的,步骤(3)中,按照所述病毒液总量计,向所述病毒液中加入质量体积百分比(m:V)0.05%~0.2%羧甲基纤维素钠,2~8℃静置过夜。
优选的,步骤(4)中,以质量体积百分比计,所述冻干保护剂包括:低聚甘露糖1%~2%、聚乙二醇2000 0.5%~1%、谷氨酸钠1%~4%、PVP K30 1%~2%,余量为去离子水。
优选的,细胞制备和病毒液制备是在二氧化碳振荡培养箱或搅拌式生物反应器中完成,所述二氧化碳振荡培养箱的培养条件为:温度37℃、5%CO2、湿度50%~80%和转速80~120rpm;所述搅拌式生物反应器的培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和转速40~50rpm。
本发明还提供一种猪瘟活疫苗,其由所述猪瘟活疫苗的制备方法制备得到。
本发明公开了以下技术效果:
本发明采用无血清全悬浮培养技术方法增殖猪瘟病毒,生产工艺易于放大、生产效率高、生产成本低,制备的成品疫苗效价高、安全性好,疫苗的保存期长,免疫效果更佳。与现有技术相比,本发明具有如下优点:
(1)通过在无血清培养基中添加适量的PEG2000、硫酸锌和PVP,增殖病毒滴度更高,且批次间更稳定,半成品毒液保存期更长。
(2)采用流加全悬浮培养方式,添加辅助液与补加悬浮细胞相结合,实现10次以上的连续收获,无需培养载体,生产规模易于放大,提高生产效率、降低生产成本。
(3)病毒纯化时,采用深层过滤方法,用羧甲基纤维素钠对毒液前处理,可有效截留杂蛋白,病毒液更纯净,制备的疫苗免疫应激小、免疫效果好。
(4)优化疫苗冻干保护剂配方,通过添加低聚甘露糖、聚乙二醇2000等成分,提升疫苗冻干保护效果,成品疫苗2~8℃保存期36个月以上,降低疫苗在储藏和运输过程中对环境的要求,且疫苗安全性更好。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包括”、“具有”等等,均为开放性的用语,即意指包含但不限于。
以下实施例中所用的实验试剂耗材,如无特殊说明,均为常规生化试剂。商品化的无血清培养基由甘肃健顺生物科技有限公司、浙江壹生科生物技术有限公司等国内厂家生产供应。
以下实施例中疫苗毒株选用猪瘟病毒兔化弱毒株(C株),猪瘟病毒兔化弱毒株(C株)活疫苗市场有售;本发明公开的制备方法不限于这个毒株,适用于该类病毒任何毒株。
以下实施例中生物反应器选用搅拌式生物反应器,本发明公开的制备方法也适用于气升式生物反应器和膜生物反应器,不同的生物反应器,生产工艺参数略有差异。
下面以具体的实施例对本发明的技术方案进一步说明。
实施例1应用ST细胞生产猪瘟病毒液
(1)细胞制备
复苏冻存的ST细胞,3000r/min离心5分钟,将细胞沉淀用无血清培养基(购自甘肃健顺生物科技有限公司)重悬,细胞密度为1.0×106个/mL,置于二氧化碳振荡培养箱培养72h,培养条件为:37℃、5%CO2、湿度50%~80%和转速80rpm,连续传代3次,全部细胞转入35L生物反应器继续放大培养72h,生物反应器的培养条件为:37.0℃、溶氧50%~90%、pH7.0~7.5和转速40rpm,细胞培养液体积23L。
(2)病毒液制备
维持液的配制:称取PEG2000 5g、硫酸锌0.3g和PVP K30 1g,溶于500mL去离子水中,用去离子水定容至1000mL,116℃灭菌40min备用,即为辅助液。无血清培养基与上述辅助液按体积比(V:V)99:1混合均匀,即为维持液。
取上述培养的20L细胞液,计数细胞密度为1.0×107个/mL,将毒种(病毒含量:107.5TCID50/mL)按MOI=0.1接种细胞液中(即加入632mL毒种),然后加入维持液180L,在500L生物反应器继续培养,生物反应器培养条件为:37.0℃、溶氧50%~90%、pH7.0~7.5和转速50rpm,培养72h收获病毒液。并设立维持液中不添加辅助液(维持液仅含无血清培养基)的对照组。
流加培养:收获时留存上述病毒液0.2L,然后补加培养好的ST细胞25L(细胞密度8.0×106个/mL,罐体培养体积200L),补加维持液175L,生物反应器培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和40rpm,37℃继续培养72h,再次收获,如此连续收获15次,共收获病毒液约3000L。
实施例2应用PK15细胞生产猪瘟病毒液
(1)细胞制备
复苏冻存的PK15细胞,3000r/min离心5min,将细胞沉淀用无血清培养基(购至浙江壹生科生物技术有限公司)重悬,细胞密度为1.0×106个/mL,置于二氧化碳振荡培养箱培养72h,培养条件为:37℃、5%CO2、湿度50%~80%和转速120rpm,连续传代3次,全部细胞转入35L生物反应器继续放大培养96h,生物反应器的培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和转速50rpm,细胞培养液体积22.5L。
(2)病毒液制备
维持液的配制:称取PEG2000 10g、硫酸锌0.1g和PVP K30 0.5g溶于500mL去离子水中,用去离子水定容至1000mL,116℃灭菌40min备用,即为辅助液。无血清培养基与上述辅助液按体积比(V:V)99:1混合均匀,即为维持液。
取上述培养的21L细胞液,计数细胞密度为1.4×107个/mL,将毒种(病毒含量:107.5TCID50/ml)按MOI=0.01接种细胞液中(即加入93mL毒种),然后加入维持液273L,在500L生物反应器继续培养,生物反应器培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和转速40rpm,培养96h收获病毒液。
流加培养:收获时留存上述病毒液3L,然后补加培养好的PK15细胞15L(细胞密度2.0×107个/mL,罐体培养体积300L),补加维持液282L,生物反应器培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和转速40rpm,37℃继续培养48h,再次收获,如此连续收获10次,共收获约3000L。
实施例3应用MPK细胞生产猪瘟病毒液
与实施例1的区别在于:MPK细胞替换ST细胞,其他步骤均相同。
实施例4病毒纯化
设置3个实验组[分别为实施例1中第1次收获(1收)、第10次收获(10收)和第15次收获(15收)的病毒液]和1个不添加辅助液的对照组,分别加入质量百分数0.05%、0.1%、0.2%和0.05%羧甲基纤维素钠,2~8℃静置过夜,然后经1~10μm深层滤板过滤,收集滤出液,置-15℃以下保存备用。
过滤前、过滤后分别取样,用市售BCA蛋白测定试剂盒测定总蛋白含量、采用间接免疫荧光法测定病毒含量。滤出液保存期试验,分别在-15℃保存2月、4月和6月时,测定病毒含量(间接免疫荧光法)。两项试验测定结果如表1所示。
表1蛋白和病毒含量测定结果
常规方法不添加辅助液,培养病毒一般可以连续收获5~8次。由上述表1可见,常规方法收获病毒液蛋白含量10mg/mL左右,病毒含量(Lg(TCID50/mL))6.5左右,-15℃保存2个月下降1个滴度以上。
本发明方法,培养病毒可以连续收获15次,收获病毒液病毒含量是常规方法的30倍,病毒液-15℃保存6个月,下降0.6个滴度以内,保存期更长,稳定性更好。
实施例5疫苗冻干与质量评价
(1)保护剂配制
保护剂1称取低聚甘露糖1g、聚乙二醇2000 1g、谷氨酸钠1g、PVP K30 2g,溶于50mL去离子水中,定容至100mL,116℃高压40min备用。
保护剂2称取低聚甘露糖2g、聚乙二醇2000 0.5g、谷氨酸钠4g、PVP K30 1g,溶于50mL去离子水中,定容至100mL,116℃高压40min备用。
(2)疫苗冻干
取实施例4中的10收、15收纯化病毒液,分别与保护剂1、保护剂2按体积比(V:V)1:1混合均匀,分装2.0mL/瓶,进行冷冻真空干燥,制成疫苗A和疫苗B。
(3)安全检验
28日龄外三元仔猪300头,随机分成3组,100头/组,分别颈部肌注试验疫苗(疫苗A、疫苗B和常规疫苗对照),2头份/头,免疫后0~7日观察精神、采食和行为等临床表现并统计。疫苗免疫仔猪临床表现情况如表2所示。
表2疫苗免疫仔猪临床表现统计结果
由表2结果可见,本发明制备的猪瘟活疫苗(疫苗A和疫苗B)分别免疫28日龄仔猪,100头/组,免后均无异常,而对照疫苗5/100出现异常反应,说明本发明制备的猪瘟活疫苗安全性更佳。
(4)效力检验
28日龄仔猪100头,随机分成4组,25头/组,1组作空白对照,其余3组分别颈部肌注试验疫苗(疫苗A、疫苗B和常规疫苗对照),1头份/头,于免疫后0月、2月采血,分离血清,用IDEXX猪瘟病毒抗体检测试剂盒进行抗体效价检测(阻断率≥40%为抗体阳性),疫苗免疫后猪抗体变化情况如表3所示。
表3疫苗免疫后猪抗体测定结果
由表3结果可见,本发明制备的猪瘟活疫苗(疫苗A和疫苗B)免疫28日龄仔猪,免后2月免疫ELISA抗体均值(不低于85%)和抗体阳性率(25/25),明显优于常规对照疫苗(58%,15/25),免疫效果更佳。
(5)疫苗保存期试验
将疫苗A和疫苗B在2~8℃冷藏保存,分别在0、12、24和36个月时取样,测定病毒含量(Lg(TCID50/头份)),并设立常规疫苗对照。保存期试验结果如表4所示。
表4保存期试验结果(Lg(TCID50/mL))
由表4结果可见,本发明制备的猪瘟活疫苗(疫苗A和疫苗B)2~8℃保存36个月仅下降0.4个滴度,明显优于常规对照疫苗(下降1个滴度以上),保存期更长,稳定性更好。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.一种猪瘟活疫苗的制备方法,其特征在于,包括以下步骤:
(1)细胞制备
用无血清培养基培养全悬浮细胞,经重悬、传代培养、扩大培养,获取细胞液;
(2)病毒液制备
将猪瘟病毒接种所述细胞液,然后补加维持液,所述维持液包括无血清培养基和辅助液,接毒后继续培养72~96h,首次收获病毒液;之后采用流加培养方式继续培养,连续培养收获10~15次病毒液;
(3)病毒纯化
所述病毒液中加入羧甲基纤维素钠,静置过夜,过滤,收集滤出液;
(4)疫苗冻干
所述滤出液中加入冻干保护剂,定量分装,真空冷冻干燥,制成猪瘟活疫苗。
2.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(1)中,所述全悬浮细胞包括ST细胞、PK15细胞和MPK细胞;全悬浮细胞重悬及传代的细胞密度均为1.0×106个/mL。
3.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(2)中,按照MOI为0.01~0.1将所述猪瘟病毒接种所述细胞液。
4.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(2)中,所述无血清培养基与所述辅助液体积比为99:1;其中,所述辅助液为PEG2000 5~10g/L、硫酸锌0.1~0.3g/L和PVP K30 0.5~1g/L的水溶液。
5.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(2)中,所述流加培养方式具体为:每次收获所述病毒液时,反应器中留存0.1~1%的病毒液,先补加培养体积比为1/20~1/8的所述细胞液,然后补加所述维持液至培养体积,37℃继续培养48~72小时,再次收获。
6.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(3)中,按照所述病毒液总量计,向所述病毒液中加入质量体积百分比(m:V)0.05%-0.2%羧甲基纤维素钠,2~8℃静置过夜。
7.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,步骤(4)中,以质量体积百分比计,所述冻干保护剂包括:低聚甘露糖1%~2%、聚乙二醇2000 0.5%~1%、谷氨酸钠1%~4%、PVP K30 1%~2%,余量为去离子水。
8.如权利要求1所述的猪瘟活疫苗的制备方法,其特征在于,细胞制备和病毒液制备是在二氧化碳振荡培养箱或搅拌式生物反应器中完成,所述二氧化碳振荡培养箱的培养条件为:温度37℃、5%CO2、湿度50%~80%和转速80~120rpm;所述搅拌式生物反应器的培养条件为:温度37.0℃、溶氧50%~90%、pH7.0~7.5和转速40~50rpm。
9.一种猪瘟活疫苗,其特征在于,其由权利要求1~8任一项所述猪瘟活疫苗的制备方法制备得到。
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