CN116200329B - 一种无血清昆虫培养基及应用 - Google Patents
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Abstract
本发明涉及一种无血清昆虫培养基及应用，属于生物制药技术领域，包括基础培养基和添加剂，所述添加剂的组分包括：葡萄糖、蛋白水解物、嘌呤类物质、PF68以及细胞因子。本发明昆虫细胞无血清培养基中添加了细胞生长因子，能够支持细胞快速增殖，并且细胞培养后期活率下降速率更慢；本发明的昆虫细胞无血清培养基组分简单明确，易于制备，可长期保存，且对其培养效果无影响，适用性广泛，既可培养Sf‑9和Sf‑21，也可以培养High‑five细胞，能够支持昆虫细胞系的稳定传代和快速增殖，能支持昆虫细胞表达多种重组蛋白。
Description
技术领域
本发明属于生物制药技术领域，具体地，涉及一种无血清昆虫培养基及应用。
背景技术
昆虫细胞培养基的发展经历了天然培养基、合成培养基和无血清培养基三个阶段。天然培养基采用取自动物体液或从组织中提取的成分作为培养液。合成培养基最大的特点是各种成分已知。无血清培养基是在已知细胞所需营养物质的基础培养基中加入适宜的促细胞生长因子，能够保证细胞生长良好无须补加血清的培养基。昆虫细胞培养基的发展主要是合成培养基的发展。目前已有三种商品化的培养基：Grace培养基、IPL41培养基和TC100培养基。现有的培养基是在分析昆虫血淋巴的化学组成基础上发展和形成的。Wyatt分析了家蚕血淋巴组分后，人工配制了一个由21种氨基酸、5种无机盐、3种有机酸、果糖、海藻糖、葡萄糖所组成的基础培养基，并相应地调节pH值和渗透压。Grace在Wyatt的昆虫培养基中加入9种水溶性的B复合维生素，改进了Wyatt的培养液，并首次建立了无脊椎动物的细胞系桉蚕蛾卵巢细胞系。
已有研究发现在合成培养基中加入动物血清(小牛血清或胎牛血清)，能更有效地促进细胞的生长与繁殖。最初的Grace培养基是将Hink液加入水解乳蛋白和酵母提取物，并用10％已灭活的胎牛血清替代了昆虫血淋巴。Gardiner等对Grace培养基进行了修改并定名为TC100，TC100适合于苜蓿丫蚊夜蛾核型多角体病毒在草地贪夜蛾细胞系(Sf细胞系)中生长，但它仅适用于实验室培养而不适用于大规模生产。Weiss等改进了最初的IPL培养基，研制了新的IPL41培养基，扩大了昆虫细胞的生产规模，有利于昆虫杆状病毒的有效生产。这三种商品化培养基既有粉末状的，又有液体的，在实验室的应用中一般是补加10％的血清用于昆虫细胞培养。但是这三种培养基并不能适合于任何细胞系，不同的昆虫细胞系可能在其中一个培养基中生长较好，因此开发适用性更为广泛、且无需添加血清的昆虫细胞培养基具有广阔的前景。
发明内容
针对背景技术中存在的技术问题，本发明的目的在于提供了一种组分种类少、制备方法简单，且能支持昆虫细胞高密度增殖及产物高表达的无血清昆虫细胞培养基，该无血清昆虫细胞培养基能够支持昆虫细胞系的稳定传代(Sf-9、Sf-21和High Five)，快速增殖和产物的高表达(最高活细胞密度：Sf-9为16.10×106cells/mL，Sf-21为13.52×106cells/mL，High-five为13.1×106cells/mL)。
为了实现上述目的，本发明采用了以下技术方案实现：
本发明的第一方面提供了一种无血清昆虫培养基，包括基础培养基和添加剂，所述添加剂包括的各组分在无血清培养基中的浓度如下：葡萄糖5-7g/L、蛋白水解物10-16g/L、嘌呤类物质10-20mg/L、PF68 1-3g/L以及细胞生长因子20-50μg/mL。
优选地，所述蛋白水解物为酵母水解物和麦芽水解物的混合物。
优选地，所述嘌呤类物质为腺嘌呤和鸟嘌呤的混合物。
优选地，所述腺嘌呤和鸟嘌呤的混合物在无血清培养基中的浓度为10-20mg/L。
优选地，所述细胞生长因子为重组人表皮细胞生长因子(EGF)、重组人碱性成纤维细胞生长因子(FGF)和重组人胰岛素样生长因子(IGF)中的任意一种或两种以上的组合。
本发明的第二方面提供了上述无血清昆虫培养基在全悬浮培养昆虫细胞中的应用。
本发明的第三方面提供了上述全悬浮培养昆虫细胞的方法，包括以下步骤：
S1、将葡萄糖、蛋白水解物、嘌呤类物质、PF68以及细胞因子加入基础培养基中，至物质全部溶解后，加入1g/L的碳酸氢钠，定容后，调节其pH至6.35-6.45，过滤除菌后，得到昆虫培养基；
S2、将昆虫细胞接种至上述昆虫培养基中，不添加血清，置于无CO2条件下，27-28℃，80-120rpm摇床培养。
优选地，步骤S2中，所述昆虫细胞为Sf-9、Sf-21或High-five细胞，
优选地，步骤S2中，所述接种时的接种量为5-10×105/mL。
本发明的有益效果：
1、本发明技术方案中，制备的无血清昆虫培养基组分简单明确，易于制备；可长期保存，对其培养效果无影响；适用性广泛，既可培养Sf-9和Sf-21，也可以培养High-five细胞；
2、本发明技术方案中，制备的无血清昆虫培养基中通过添加细胞生长因子，能够支持昆虫细胞系的稳定传代和快速增殖(最高活细胞密度：Sf-9为16.10×106cells/mL，Sf-21为13.52×106cells/mL，High-five为13.1×106cells/mL)和多种重组蛋白的高表达，适用于昆虫细胞的大规模培养和应用。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
一、实验用试剂：基础培养基为Grace’s Insect Medium；葡萄糖、谷氨酰胺、碳酸氢钠、腺嘌呤、鸟嘌呤均来自sigma；酵母水解物和麦芽水解物来自Gibco。对照组培养基为商品化培养HyQSFX-InsectSFM，来源于壹生科。
二、无血清昆虫细胞培养基的各组分含量的配方具体见下表1。
表1
三、配制方法
(a)Grace’s Insect Medium配制：具体配方见表2。
表2
(b)配制：按照表1配方，向上述Grace’s Insect Medium中加入葡萄糖、酵母水解物、麦芽水解物、PF68、腺嘌呤、鸟嘌呤和细胞生长因子，待全部物质完全溶解后再加1g/L碳酸氢钠后充分混匀，定容至10L，调节pH值至6.35，囊氏滤器过滤除菌，在2-8℃保存备用。
四、昆虫细胞的悬浮培养及重组蛋白的表达
(a)细胞复苏：用对照培养基HyQSFX-InsectSFM按常规方法从液氮罐中取出冻存的细胞，迅速放入37℃水浴锅中解冻后，于超净台中进行复苏。复苏后的Sf-9、Sf-21和High-five细胞，置27℃100rpm摇床培养。
(b)细胞培养：每一种细胞按适当的密度分别用实施例1-4和对照培养基HyQSFX-InsectSFM接种培养Sf-9、Sf-21和High-five细胞，不添加血清，置27℃，100rpm摇床，每24h计数细胞密度和细胞活率。结果详见表3-5。
表3为实施例1-4和及对照组的培养基培养的Sf-9细胞的细胞密度和细胞活力
表4为实施例1-4及对照组培养基培养的Sf-21细胞的细胞密度和细胞活力
表5为实施例1-4及对照组培养基培养的High-five细胞的细胞密度和细胞活力
由上述表3-5结果可知，采用本发明实施例1-3无血清培养基和对照培养基HyQSFX-InsectSFM分别培养Sf-9、Sf-21和High-five细胞，细胞均能够得到很好的全悬浮生长，且采用本发明实施例1-3无血清培养基培养的三种细胞的最大增殖浓度均高于对照组培养基HyQSFX-InsectSFM。这是由于本发明实施例1-3昆虫细胞无血清培养基中添加了细胞生长因子，能够支持细胞快速增殖，并且能够提高细胞在培养后期的细胞活率。与其他组别对比，实施例4昆虫细胞无血清培养基培养的细胞密度有少许降低，并且细胞培养后期活率下降的比其他组别更快，这是由于实施例4中未添加细胞生长因子；由上述实验结果可得到以下结论：本发明昆虫细胞无血清培养基能够支持昆虫细胞系的稳定传代和快速增殖(最高活细胞密度：Sf-9为16.10×106cells/mL，Sf-21为13.52×106cells/mL，High-five为13.1×106cells/mL)。
(c)将实施例2中配制的无血清昆虫细胞培养基分别在不同时间点制备批次粉末培养基，使之保存时间分别为6个月及12个月，再将两批次的培养基用于培养Sf-9、Sf-21和High-five细胞，不添加血清，置27℃，100rpm摇床，每24h计数细胞密度和细胞活力，评价保存时间对培养基效果的影响。结果详见表6-8。
表6为不同保存时间的培养基培养的Sf-9细胞的细胞密度和细胞活力
表7为不同保存时间的培养基培养的Sf-21细胞的细胞密度和细胞活力
表8为不同保存时间的培养基培养的High-five细胞的细胞密度和细胞活力
由上述表6-8结果可知，本发明实施例2中配制的无血清昆虫细胞培养基可长期保存，对其培养效果无影响，且适用性广泛，既可培养Sf-9、Sf-21，也可以培养High-five细胞，上述三种细胞均能够得到很好的全悬浮生长。
(d)重组杆状病毒接种及重组蛋白的表达：按照实施例2的配方配制培养基，培养昆虫细胞Sf-9、Sf-21和High-five取细胞数量达到2×106/mL，且细胞活力在95％以上，按照0.1％V/V的接种重组杆状病毒。将接毒后的细胞置于27℃，110rpm摇床培养，每24h对细胞进行计数，并观察细胞病变情况，当90％以上的细胞发生病变，细胞活力降低，病变细胞体积明显大于正常细胞，收集培养液，1000rpm，离心10min，收集上清，Western blot实验对蛋白进行定量。结果见表9。
表9.实施例2培养基与对照组培养基培养昆虫细胞接种杆状病毒表达不同重组蛋白的灰度比值
由上述表9结果可知，本发明实施例2培养基培养昆虫细胞接种杆状病毒能够支持多种重组蛋白(PCV2 Cap、猪瘟E2蛋白和兔瘟P60蛋白)的高表达，且实施例中蛋白表达量均高于对照组。
以上内容仅仅是对本发明所作的举例和说明，所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，只要不偏离发明或者超越本权利要求书所定义的范围，均应属于本发明的保护范围。
Claims (5)
1.一种无血清昆虫培养基，其特征在于，包括基础培养基和添加剂，所述添加剂包括的各组分在无血清培养基中的浓度如下：葡萄糖5-7g/L、蛋白水解物10-16g/L、嘌呤类物质10-20mg/L、PF68 1-3g/L以及细胞生长因子20-50mg/L；
所述蛋白水解物为酵母水解物和麦芽水解物的混合物；
所述嘌呤类物质为腺嘌呤和鸟嘌呤的混合物；
所述腺嘌呤和鸟嘌呤的混合物在无血清昆虫培养基中的浓度为10-20mg/L；
所述细胞生长因子为EGF、FGF和IGF中的一种或两种以上的组合；
所述基础培养基为Grace’s Insect Medium培养基。
2.如权利要求1所述的无血清昆虫培养基在全悬浮培养昆虫细胞中的应用。
3.一种利用权利要求1所述的无血清昆虫培养基进行全悬浮培养昆虫细胞的方法，其特征在于，包括以下步骤：
S1、将葡萄糖、蛋白水解物、嘌呤类物质、PF68以及细胞生长因子加入基础培养基中，至物质全部溶解后，加入1g/L的碳酸氢钠，定容后，调节其pH至6.35-6.45，过滤除菌，得到无血清昆虫培养基备用；
S2、将昆虫细胞接种至上述无血清昆虫培养基中，不添加血清，置于无CO2条件下，27-28℃，80-120rpm摇床培养。
4.根据权利要求3所述的全悬浮培养昆虫细胞的方法，其特征在于，步骤S2中，所述昆虫细胞为Sf-9、Sf-21或者High-five细胞。
5.根据权利要求3所述的全悬浮培养昆虫细胞的方法，其特征在于，步骤S2中，所述接种时的接种量为5-10×105/mL。
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