CN113151183A - 一种促进蛋白表达的培养基添加物及其应用 - Google Patents
一种促进蛋白表达的培养基添加物及其应用 Download PDFInfo
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Abstract
本发明公开了一种促进蛋白表达的培养基添加物，属于细胞培养技术领域。包括：维生素C、乙醇胺、L‑天冬酰胺、L‑盐酸半胱氨酸、L‑胱氨酸二盐酸盐、L‑脯氨酸、L‑色氨酸、大豆蛋白胨、N‑乙酰半胱氨酸和普朗尼克F68。将促进蛋白表达的培养基添加物加入DMEM、DMEM/F12、RPMI1640基础培养基，可使细胞表达蛋白产量增加，为提升蛋白类抗体药物打下基础。
Description
技术领域
本发明属于细胞培养技术领域，具体涉及一种促进蛋白表达的培养基添加物及其应用。
背景技术
蛋白表达是指用模式生物如细菌、酵母、动物细胞或者植物细胞表达外源基因蛋白的一种分子生物学技术。在基因工程技术中占有核心地位。
哺乳动物表达系统在蛋白的起始信号、加工、分泌、糖基化方面具有独特优势，适合表达完整的大分子蛋白。由哺乳动物细胞翻译后再加工修饰产生的外源蛋白质，在活性方面远胜于原核表达系统及酵母、昆虫细胞等真核表达系统，更接近于天然蛋白质，但构成复杂、操作技术要求高、表达产量不大、产率低，且有时会导致病毒感染等是该表达系统的不足之处。
因此，如何提供一种促进蛋白表达的培养基添加物及其应用方法是本领域亟待解决的问题。
发明内容
本发明公开了一种促进蛋白表达的培养基添加物及其应用。
针对以上问题，本发明研制培养基添加物可以在基础培养基之上，做到增加蛋白产量的需求。
为了实现上述目的，本发明采用如下技术方案：
一种促进蛋白表达的培养基添加物，包括：维生素C、乙醇胺、L-天冬酰胺、L-盐酸半胱氨酸、L-胱氨酸二盐酸盐、L-脯氨酸、L-色氨酸、大豆蛋白胨、N-乙酰半胱氨酸、普朗尼克；
更优选的技术方案，终浓度为：维生素C10-50μM、乙醇胺10-50μM、L-天冬酰胺10-50mg/L、L-盐酸半胱氨酸5-50mg/L、L-胱氨酸二盐酸盐10-50mg/L、L-脯氨酸20-60mg/L、L-色氨酸10-50mg/L、大豆蛋白胨1-5g/L、N-乙酰半胱氨酸2-10mM、普朗尼克F681-5g/L。
更优选的技术方案，终浓度为：维生素C 20μM、乙醇胺25μM、L-天冬酰胺30mg/L、L-盐酸半胱氨酸30mg/L、L-胱氨酸二盐酸盐35mg/L、L-脯氨酸35mg/L、L-色氨酸25mg/L、大豆蛋白胨2g/L、N-乙酰半胱氨酸5mM、普朗尼克F681.5g/L。
上述促进蛋白表达的培养基添加物在制备细胞表达蛋白产物培养基中的应用；
一种含有促进蛋白表达的培养基添加物的蛋白表达的培养基，基础培养基为DMEM、DMEM/F12、RPMI1640培养基；
一种含有促进蛋白表达的培养基添加物的蛋白表达的培养基，其特征在于，所述培养基培养细胞种类包括：CHO细胞、BHK-21细胞、293细胞、杂交瘤细胞、Vero细胞和MDCK细胞；
一种利用蛋白表达培养基的蛋白表达培养方法，宿主细胞包括：CHO细胞、BHK-21细胞、293细胞；
更优选的技术方案，宿主细胞为悬浮培养；
更优选的技术方案，在蛋白表达环节需要去除培养基中的血清成分。
综上所述，发明公开了一种促进蛋白表达的培养基添加物，包括：维生素C、乙醇胺、L-天冬酰胺、L-盐酸半胱氨酸、L-胱氨酸二盐酸盐、L-脯氨酸、L-色氨酸、大豆蛋白胨、N-乙酰半胱氨酸和普朗尼克F68。将促进蛋白表达的培养基添加物加入DMEM、DMEM/F12、RPMI1640基础培养基，可使细胞表达蛋白产量增加，与常规培养基相比，蛋白表达增产量可达75％。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本发明表达载体mCherry2-N1的结构图。
具体实施方式
下面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做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
蛋白表达方法为本领域常规方法，具体如下：
1.使用前，将所有试剂充分混匀。不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。
2.根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。
3加入稀释好后的标准品50ul于反应孔、加入待测样品50ul于反应孔内。立即加入50ul的生物素标记的抗体。盖上膜板，轻轻振荡混匀，37℃温育45分钟。
4.甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作4次。如果用洗板机洗涤，洗涤次数增加一次。
5.每孔加入100ul的亲和链酶素-HRP，轻轻振荡混匀，37℃温育30分钟。
6.甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作4次。如果用洗板机洗涤，洗涤次数增加一次。
7.每孔加入底物A、B各50ul，轻轻振荡混匀，37℃温育5分钟。避免光照。
8.取出酶标板，迅速加入50ul终止液，加入终止液后应立即测定结果。
9.在450nm波长处测定各孔的OD值。
表达载体为常规表达载体，具体如下：
表达载体为mCherry2-N1(如图1所示)，将RRVIII(凝血因子III)基因通过Nhe1和HindIII酶切位点连入载体，构建融合表达载体，备用。
实施例1
对照组培养基：商品化培养基
302培养(Millipore Sigma,24571C)。
试验组培养基：在DMEM/F12基础培养基上再加入添加物，添加物成分和工作浓度：维生素C10μM、乙醇胺10μM、L-天冬酰胺10mg/L、L-盐酸半胱氨酸5mg/L、L-胱氨酸二盐酸盐10mg/L、L-脯氨酸20mg/L、L-色氨酸10mg/L、大豆蛋白胨1g/L、N-乙酰半胱氨酸2mM、普朗尼克F681g/L。
分别将悬浮BHK-21细胞在对照组培养基和实验组培养基中培养，在康宁125摇瓶中，37℃，8％CO2，摇床上130rpm培养，当细胞密度达到2×106个/ml时，可进行传代，传代比例1:4。当细胞密度达到2×106个/ml时，进行细胞质粒转染，将构建好的融合表达载体浓度按照每18μg的质粒DNA转染1.5×107个细胞的比例稀释到30ml的Gibcoopti-MEM培养液中的BHK-21细胞，之后运用电转仪进行转染。收集细胞，将电击池放入电穿孔装置的架上用所设定的电压及电容值电击一次，之后将电击池置于冰上10min；分别用对应的对照组培养基和实验组培养基20倍稀释被转染的细胞，并用该液洗电击池以移出所有的被转染细胞。培养细胞50-60h后，收集细胞进行蛋白表达分析。结果如表1所示。
实施例2
对照组培养基：CHO细胞、BHK-21细胞和293细胞在商品化培养基
302培养(Millipore Sigma,24571C)。
试验组培养基：在DMEM/F12基础培养基上再加入添加物，添加物成分和工作浓度：维生素C 50μM、乙醇胺50μM、L-天冬酰胺50mg/L、L-盐酸半胱氨酸50mg/L、L-胱氨酸二盐酸盐50mg/L、L-脯氨酸60mg/L、L-色氨酸50mg/L、大豆蛋白胨5g/L、N-乙酰半胱氨酸10mM、普朗尼克F685g/L。
分别将悬浮BHK-21细胞在对照组培养基和实验组培养基中培养，在康宁125摇瓶中，37℃，8％CO2，摇床上130rpm培养，当细胞密度达到2×106个/ml时，可进行传代，传代比例1:4。当细胞密度达到2×106个/ml时，进行细胞质粒转染，将构建好的融合表达载体浓度按照每18μg的质粒DNA转染1.5×107个细胞的比例稀释到30ml的Gibco opti-MEM培养液中的BHK-21细胞，之后运用电转仪进行转染。收集细胞，将电击池放入电穿孔装置的架上用所设定的电压及电容值电击一次，之后将电击池置于冰上10min；分别用对应的对照组培养基和实验组培养基20倍稀释被转染的细胞，并用该液洗电击池以移出所有的被转染细胞。培养细胞50-60h后，收集细胞进行蛋白表达分析。结果如表1所示。
实施例3
对照组培养基：商品化培养基
302培养(Millipore Sigma,24571C)。
试验组培养基：在DMEM/F12基础培养基上再加入添加物，添加物成分和工作浓度：维生素C 20μM、乙醇胺25μM、L-天冬酰胺30mg/L、L-盐酸半胱氨酸30mg/L、L-胱氨酸二盐酸盐35mg/L、L-脯氨酸35mg/L、L-色氨酸25mg/L、大豆蛋白胨2g/L、N-乙酰半胱氨酸5mM、普朗尼克F681.5g/L。
分别将悬浮BHK-21细胞在对照组培养基和实验组培养基中培养，在康宁125摇瓶中，37℃，8％CO2，摇床上130rpm培养，当细胞密度达到2×106个/ml时，可进行传代，传代比例1:4。当细胞密度达到2×106个/ml时，进行细胞质粒转染，将构建好的融合表达载体浓度按照每18μg的质粒DNA转染1.5×107个细胞的比例稀释到30ml的Gibco opti-MEM培养液中的BHK-21细胞，之后运用电转仪进行转染。收集细胞，将电击池放入电穿孔装置的架上用所设定的电压及电容值电击一次，之后将电击池置于冰上10min；分别用对应的对照组培养基和实验组培养基20倍稀释被转染的细胞，并用该液洗电击池以移出所有的被转染细胞。培养细胞50-60h后，收集细胞进行蛋白表达分析。结果如表1所示。
表1
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对上述实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一种促进蛋白表达的培养基添加物，其特征在于，包括：维生素C、乙醇胺、L-天冬酰胺、L-盐酸半胱氨酸、L-胱氨酸二盐酸盐、L-脯氨酸、L-色氨酸、大豆蛋白胨、N-乙酰半胱氨酸、普朗尼克。
2.如权利要求1所述的一种促进蛋白表达的培养基添加物，其特征在于，终浓度为：维生素C 10-50μM、乙醇胺10-50μM、L-天冬酰胺10-50mg/L、L-盐酸半胱氨酸5-50mg/L、L-胱氨酸二盐酸盐10-50mg/L、L-脯氨酸20-60mg/L、L-色氨酸10-50mg/L、大豆蛋白胨1-5g/L、N-乙酰半胱氨酸2-10mM、普朗尼克F681-5g/L。
3.如权利要求2所述的一种促进蛋白表达的培养基添加物，其特征在于，终浓度为：维生素C 20μM、乙醇胺25μM、L-天冬酰胺30mg/L、L-盐酸半胱氨酸30mg/L、L-胱氨酸二盐酸盐35mg/L、L-脯氨酸35mg/L、L-色氨酸25mg/L、大豆蛋白胨2g/L、N-乙酰半胱氨酸5mM、普朗尼克F681.5g/L。
4.如权利要求1-3任一所述的促进蛋白表达的培养基添加物在制备细胞表达蛋白产物培养基中的应用。
5.一种含有如权利要求1-3任一所述的促进蛋白表达的培养基添加物的蛋白表达培养基，其特征在于，基础培养基为DMEM、DMEM/F12、RPMI1640培养基。
6.一种利用权利要求5所述的蛋白表达培养基添加物的蛋白表达培养方法，其特征在于，宿主细胞种类包括：CHO细胞、BHK-21细胞、293细胞。
7.一种如权利要求6所述的蛋白表达培养方法，其特征在于，宿主细胞为悬浮培养。
8.一种如权利要求6所述的蛋白表达培养方法，其特征在于，在蛋白表达环节需要去除培养基中的血清成分。
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