CN1060110A - 制备口服用活伤寒疫苗的培养方法 - Google Patents

制备口服用活伤寒疫苗的培养方法 Download PDF

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CN1060110A
CN1060110A CN91108798A CN91108798A CN1060110A CN 1060110 A CN1060110 A CN 1060110A CN 91108798 A CN91108798 A CN 91108798A CN 91108798 A CN91108798 A CN 91108798A CN 1060110 A CN1060110 A CN 1060110A
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朴凤和
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Abstract

一种获得口服用活伤寒疫苗的培养方法，包括下 述步骤：在BHI培养基中培养得自伤寒菌株Ty21a 的种子细菌，将所得的培养细菌悬浮于保护性介质中 并在安瓿瓶中将所述悬浮细菌冻干，使冻干的细菌复 活并将复活的细菌接种于生产培养基中，在发酵罐中 培养细菌，以及将细菌冻干并配成口服用的制剂。

Description

本发明涉及一种制备口服用的活伤寒疫苗的培养方法，更具体地说，涉及一种利用新的生长培养基、保护性介质并控制培养温度以在培养后获得更多的细菌细胞和在冰冻干燥后获得更高存活率的具免疫活性的减毒活细菌的培养方法。
已知伤寒的病菌是伤寒沙门菌（Salmonella  typhi），伤寒通过污染的水或食物、拥挤区域中人与人接触而感染。
为了防止伤寒，通常使用非口服性死疫苗或口服性活疫苗。
现在，对口服疫苗的使用逐渐增加，因为死疫苗虽然很安全，但没有所需的致免疫活性。
制备活疫苗的方法不同于制备死疫苗的方法。制备活疫苗的已知方法如下：
ⅰ）在液体或固体培养基中培养伤寒沙门杆菌的减毒突变性，ⅱ）离心并冰冻干燥，和ⅲ）配成制剂;而死疫苗的制备方法如下：ⅰ）在液体或固体培养基培养伤寒细菌，ⅱ）用丙酮、苯酚或福尔马林灭菌，和ⅲ）配成制剂。
为了得到活疫苗，通常使用减毒突变体伤寒沙门菌Ty21a，该突变体已由R.Germanier和E.Furer从伤寒沙门菌Ty2经两步突变处理而分离。该突变体的特征是缺少尿苷二核酸半乳糖-4-差向异构酶的活性，该酶是形成细菌细胞壁脂多糖的必要中间体。因此，这种突变体实际上没有毒性，因为它只有不完整的细胞壁。
然而，这种突变体在半乳糖的存在下通过合成脂多糖而表现出杰出的免疫效应。且过量提供脂多糖会引起导致溶菌作用的半乳糖-1-磷酸酯和UDP-半乳糖的积累。
R.Germanier等人获得了关于Ty21a菌株的突变、培养和配方的专利US  3，856，935，1972年12月24日公开。
以下是该专利中所公开的制备伤寒疫苗的方法。
将选择的突变体Ty21a在摇床上在脑心浸液中37℃下培养6小时。所得细菌通过在6，000g下离心而收集，然后悬浮于含有8%蔗糖、1.5%明胶和5%脱脂乳粉的保护性水介质中。将悬浮液在小瓶中冰冻干燥后，打开一个小瓶制备细菌，并接种到一营养琼脂斜面上，然后将该斜面在37℃培养。将细菌悬浮于生理盐水溶液中并将该悬浮液作为菌种接种到600ml营养液中。所述营养液如下制备：将28g酪蛋白水解物、10g酵母提取物和2g葡萄糖溶于1升蒸馏水中并将pH调至7.2。将所述培养物转移到-25升的同样营养液中，接种后在37℃下培养12小时，同时以每分钟5升空气的速率通气。培养后，通过在6，000g下离心而收集细菌细胞，然后悬浮于上述保护性水介质中并在小瓶中冰冻干燥。
但是，按照上述方法培养出的细菌在冰冻干燥之后存活率很低，因为Ty21a菌株对环境改变的抗性较弱，故在冰冻干燥期间易被杀死。
本发明的目的是提供一种改进的培养方法，该方法利用新的生长介质、保护性介质和控制的培养温度以在培养后获得更多的培养细胞及在冰冻干燥后获得高存活率的具有免疫活性的减毒活细菌。本发明设计的生长介质可以通过控制培养温度使细菌有效地生长并容易适应环境的改变。本发明设计的保护介质可以使细菌在冰冻干燥期间耐受高速冰冻，并且冻干的细菌可以贮存较长的时间而不改变其性质。按照本发明的培养方法利用新的生长介质和保护介质获得的口服伤寒疫苗表现出减毒细菌的高存活率和抗伤寒的高免疫活性。
本发明的详细培养方法叙述如下。
用于制备口服伤寒疫苗的伤寒菌株是伤寒沙门杆菌的减毒突变株Ty21a，该菌株已存藏于美国典型培养物保藏中心（ATCC，保藏号为ATCC-33459）和朝鲜科学技术研究所（KIST，保藏号为KCTC-2425）。上述保藏的伤寒菌株也可通过使商品“Vivotif  Berna”〔marketed  by  Swiss  Serus  ＆  Vaccine  Institute  Berna〕以单菌落的形式在由脑心浸液琼脂构成的再生介质中再生而获得。
将从上述伤寒菌株的单集落中分离出的种子细菌接种于100ml脑心浸液（“BHI”）介质中，并将接种的菌株在摇床上30℃下培养18小时。将培养物在6，000g下离心10分钟而收集细菌，并悬浮于保护介质中。所述保护介质含有8%乳糖、1%羧甲基纤维素、5%脱脂奶、0.2%MgSO47H2O和1%谷氨酸一钠。
将上述悬浮液在安瓿瓶中冰冻干燥。在制备活疫苗前将上述冻干的安瓿瓶存于4-8℃下。
打开安瓿瓶得到细菌并将其接种于BHI培养基上。将接种的细菌在摇床上30℃下培养18小时以培养种子细菌。将得到的种子细菌接种到生长培养基中进行主培养，所述生长培养基含有37g BHI、5g L-赖氨酸、30g 山梨糖醇、1.5g K2HPO4、0.5g MgSO47H2O和1升蒸馏水，pH为7.0。在大量生产的情况下，可将得自生产培养基的培养物接种到250升的发酵罐中。在主培养期间，必须改变作为本发明主要特征之一的培养温度，以使细菌充分适应冰冻干燥期间的快速温度变化。主培养期间的培养温度为ⅰ）头6个小时30℃，ⅱ）接下来的2个小时25℃，和ⅲ）最后14小时20℃。发酵罐的通气量维持在0.5VVm（Volume/Volume/minute）。
培养完成后，在6，000g下离心而收集细胞，并在0-4℃下在上述保护性介质中悬浮2小时。将该悬浮液在冻干器上冰冻干燥。最后，将冻干的细菌与乳糖混合以使每克中含有2.5×1010个活细菌，然后将制得的疫苗装入胶囊中，0.2g/胶囊，制得口服用的胶囊。
本发明可在下面的实施例中更详细地说明。但这些实施例仅仅是说明性的，不可视为对本发明范围的限制。
实例1
将得自ATCC-33459、KCTC-2425或Vivotif Berna的减毒突变菌株伤寒沙门菌Ty21a接种于100ml BHI培养基中，并在摇床上30℃下培养18小时，然后将离心收集的细菌在安瓿瓶中冰冻干燥。将作为菌种的冻干细菌接种到主生长培养基中，所述主生长培养基由37g BHI、5g L-赖氨酸、30g 山梨醇、1.5g K2HPO4、0.5g MgSO47H2O和1升蒸馏水组成，pH为7.0。
为了进行主培养，将5升主生产培养基转移到10升的发酵罐中。发酵罐的通气速率为0.5VVm，搅拌为300rpm。主培养期间的温度为ⅰ）头6小时30℃，ⅱ）接下来的2小时25℃和ⅲ）最后14小时20℃。
整个培养时间为22小时。最终得到的培养液的性质如下：培养液浊度为OD 660下0.516×20，活细菌数为4.6×1010/ml。
实例2
用与实例1同样的方式进行培养，只是主培养温度为ⅰ）头6-8个小时30℃，ⅱ）接下来的2-4小时25℃和ⅲ）最后12-14小时20-25℃。
最终得到的培养液的性质如下：浊度为OD 660下0.452×20-0.516×20，活细菌数目为3.2×1010/ml。
实例3
将得自实例1的培养液在6，000g下离心，并悬浮于2.5升保护性介质中，所述保护性介质由8%乳糖、1%羧甲基纤维素、5%脱脂奶、0.2%MgSO47H2O、1%谷氨酸-钠组成。将所述在保护介质中的悬浮液在0-4℃下放置2小时以增加对低温的抗性。将抗性增加的悬浮液在冻干器中冰冻2小时并在真空状态下干燥12小时。
冰冻干燥后的活细菌数目为6.8×1010/g。
实例4
用1%明胶代替1%羧甲基纤维素重复实例3的冰冻干燥过程。冰冻干燥后得到活细菌为4.1×1010/克。

Claims (7)

1、一种获得口服用活伤寒疫苗的培养方法，它包括：
ⅰ)在BHI培养基中培养得自伤寒菌株Ty21a的种子细菌，
ⅱ)将培养的细菌悬浮于保护介质中，并在安瓿瓶中冰冻干燥，
ⅲ)使冻干的细菌复活，并将复活的细菌接种于生产培养基中，
ⅳ)在发酵罐中培养细菌，和
ⅴ)将所述细菌进行冰冻干燥并配成口服用的制剂。
2、根据权利要求1的培养方法，其中所述保护性介质由8%乳糖、1%羧甲基纤维素、5%脱脂奶、0.2% MgSO47H2O和1%谷氨酸-钠组成。
3、根据权利要求1的培养方法，其中生长培养基由37g BHI、5g L-赖氨酸、30g 山梨醇、1.5g K2HPO4、0.5g MgSO4和1升蒸馏水（pH调至7.0）组成。
4、根据权利要求1-3中的一项培养方法，其中发酵罐的培养温度为ⅰ）头6个小时30℃，ⅱ）接下来的2小时25℃和ⅲ）最后14小时20℃。
5、根据权利要求2的培养方法，其中保护介质中的1%羧甲基纤维素由1%明胶代替。
6、根据权利要求1的培养方法，其中所述悬浮液在0-4℃下放置2小时后，对所述细菌进行冰冻干燥，在-70℃下冰冻2小时，在真空下干燥12小时。
7、根据上述权利要求中一项所述的培养方法得到的活伤寒疫苗。
CN91108798A 1990-09-05 1991-09-05 制备口服用活伤寒疫苗的培养方法 Expired - Lifetime CN1056876C (zh)

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CN105663171A (zh) * 2009-01-09 2016-06-15 剑桥企业有限公司 用于口服递送的活细胞制剂

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CN105663171A (zh) * 2009-01-09 2016-06-15 剑桥企业有限公司 用于口服递送的活细胞制剂
CN105663171B (zh) * 2009-01-09 2020-09-22 剑桥企业有限公司 用于口服递送的活细胞制剂
CN104066834A (zh) * 2011-12-22 2014-09-24 万科斯蒙股份有限公司 产生高产的沙门氏菌减毒菌株的方法
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GB9117918D0 (en) 1991-10-09
GB2248241A (en) 1992-04-01
JPH0771471B2 (ja) 1995-08-02
ES2037599A1 (es) 1993-06-16
KR920006496A (ko) 1992-04-27
CN1056876C (zh) 2000-09-27
CH683187A5 (fr) 1994-01-31
ES2037599B1 (es) 1994-02-01
IL99138A0 (en) 1992-07-15

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