CN116478913B - 一种cho细胞培养基及其应用 - Google Patents
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Abstract
本发明涉及一种CHO细胞培养基,包括基础培养基和添加剂,本发明的CHO细胞培养基可以增强CHO细胞瞬时转染表达蛋白,通过添加组合物,补料的方式,能在细胞转染后,维持高活率,使培养周期延长,从而提高蛋白的表达量。本发明含有多种高浓度氨基酸和一些其他核苷酸及特殊成分,其中白藜芦醇、丁酸钠、丙戊酸、诺考达唑、普鲁卡因胺、肼苯哒嗪的联合作用,可显著提高蛋白产量,且不影响细胞的正常培养,高浓度氨基酸的添加,尤其L‑半胱氨酸和酪氨酸的添加,可延长细胞存活的时间,从而使蛋白表达进一步提高,长达13‑14天的培养,使蛋白表达提高1.4‑2.24倍。
Description
技术领域
本发明涉及细胞培养基领域,具体为一种CHO细胞培养基及其应用。
背景技术
重组蛋白的生产方法有稳定转染和瞬时转染,随着生物药技术的发展,CHO细胞稳定转染的蛋白产量已达到3-10g/L,非常可观,但是稳转细胞株开发周期长,仍然有大量对于瞬转的需求,以缩短产品开发周期,短期内快速获得所需蛋白和质量评估反馈,而瞬转表达蛋白的关键因素有很多,如细胞系、表达载体、质粒DNA质量、培养基、转染方法及转染试剂等。
其中细胞系的选择很有限,一般是CHO和HEK293,HEK293容易转染,受到广泛应用,而CHO细胞因其转染难度较大,表达量偏低,难以放大等因素而受限,近年来,Expi CHO表达体系的出现,颠覆了传统认知,在Expi CHO-S细胞中,不仅保留了悬浮细胞高密度生长的特性,其瞬转的表达量可以达到3g/L以上,但其转染试剂为脂质体,虽然转染效率和蛋白表达均有显著提高,但价格较高,未能广泛应用,其他的转染方式,也因为各种各样的原因,限制了其大规模工业化生产,目前研究最多的阳离子聚合物,不仅价格低廉,且转染效率相对较高,但目前应用较多的PEI,其缺点是随着PEI用量的增加,毒性也随之增加,想要用少量的PEI,获得较高的转染效率,或者用其他阳离子聚合物替代PEI,成为大家研究的焦点。
大量的研究表明,想要提高CHO细胞瞬转的蛋白表达,不能单一地优化某个关键因素,要进行DoE设计,找到最优的实验方案,如用转染方法,转染细胞密度、DNA和转染试剂的比例、孵育时间、培养基的选择等,均需进行进一步优化,而其中培养基的选择,不仅影响转染效率,更是蛋白最终产量的最关键因素,但是由于细胞对培养基需求不同,培养基通用性不足,如何选择是个难题,通过添加组合物,补料的方式,能在细胞转染后,维持高活率,使培养周期延长,从而提高蛋白的表达量。
发明内容
针对现有技术的不足,本发明提供一种CHO细胞培养基,用于增强CHO细胞瞬时转染表达蛋白的组合物,提高蛋白表达,以弥补现有技术的不足。
为了解决上述技术问题,本发明提供如下技术方案:
一种CHO细胞培养基,包括基础培养基和添加剂,所述的基础培养基为DMEM/F12培养基,所述的添加剂为:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02~0.04g/L、尿苷0.02~0.04g/L、鸟苷0.02g~0.04/L、甘露聚糖7g/L、白藜芦醇0.1~0.5g/L、丁酸钠0.16~0.8g/L、丙戊酸0.16~0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1~1g/L、普鲁卡因胺0.1~1g/L、肼苯哒嗪0.1~0.5g/L。
优选的,所述的添加剂为:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
优选的,所述的CHO细胞为CHO-S细胞。
优选的,所述的CHO-S细胞为Expi CHO-S细胞。
一种前述CHO细胞培养基的应用,用于CHO细胞的培养。
与现有技术相比,本发明的有益效果如下:
本发明提出了一种增强CHO细胞瞬时转染表达蛋白的培养基及其应用,搭配市售CHO瞬转基础培养基DMEM/F12培养基,通过添加组合物,补料的方式,能在细胞转染后,维持高活率,使培养周期延长,从而提高蛋白的表达量。本发明含有多种高浓度氨基酸和一些其他核苷酸及特殊成分,其中白藜芦醇、丁酸钠、丙戊酸、诺考达唑、普鲁卡因胺、肼苯哒嗪的联合作用,可显著提高蛋白产量,且不影响细胞的正常培养,高浓度氨基酸的添加,尤其L-半胱氨酸和酪氨酸的添加,可延长细胞存活的时间,从而使蛋白表达进一步提高,长达13-14天的培养,使蛋白表达提高1.4-2.24倍。
本发明的作用机理如下:
丁酸钠、丙戊酸、普鲁卡因胺、肼苯哒嗪等均为常见的DNMT(DNA甲基转移酶)抑制剂和HDAC(组蛋白去乙酰化酶)抑制剂,一方面可以作为HDAC竞争性抑制剂,与酶的催化中心结合,另一方面可以促进HDAC2泛素依赖的蛋白酶体降解,通过解除转录抑制来提高重组蛋白表达量。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1为对照组与不同实验组的活细胞密度曲线图;
图2为对照组与不同实验组的细胞活率曲线图;
图3为对照组和实施例1/4/5/6的不同抗体表达柱状图;
图4为对照组在24小时的转染效率示意图;
图5为添加实施例1中上述组合物在24小时的转染效率示意图;
图6为对照组在48小时的转染效率示意图;
图7为添加实施例1中上述组合物在48小时的转染效率示意图。
具体实施方式
下面将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。
一、实验试剂:
以下实施例中所述的添加剂,均采购自Sigma;商业化培养基DMEM/F-12(货号:11320033)、DMEM(货号:11995)、Expi CHO基础培养基、Expi CHO转染试剂盒(含Expi CHO补料培养基)(货号A29129)均采购自赛默飞(Gibco);细胞株Expi CHO-S采购自赛默飞(Gibco)、质粒购自苏州金唯智生物科技有限公司;转染试剂购自安诺伦(北京)生物科技有限公司。
二、实验方法:
实施例1:添加剂具体包括:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
实施例2:添加剂具体包括:L-天冬氨酸10.56g/L、L-天冬酰胺10.56g/L、L-精氨酸6.88g/L、L-组氨酸8g/L、L-异亮氨酸4g/L、L-亮氨酸4g/L、L-赖氨酸10.56g/L、L-蛋氨酸6.4g/L、L-苯丙氨酸4.4g/L、L-苏氨酸16g/L、L-色氨酸2.24g/L、L-酪氨酸12g/L、L-缬氨酸10.56g/L、L-半胱氨酸3.52g/L、L-谷氨酸4.4g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
实施例3:添加剂具体包括:L-天冬氨酸15.84g/L、L-天冬酰胺15.84g/L、L-精氨酸10.32g/L、L-组氨酸12g/L、L-异亮氨酸6g/L、L-亮氨酸6g/L、L-赖氨酸15.84g/L、L-蛋氨酸9.6g/L、L-苯丙氨酸6.6g/L、L-苏氨酸24g/L、L-色氨酸3.36g/L、L-酪氨酸18g/L、L-缬氨酸15.84g/L、L-半胱氨酸5.28g/L、L-谷氨酸6.6g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖25g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
实施例4:添加剂具体包括:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑1.0g/L、普鲁卡因胺1.0g/L、肼苯哒嗪0.5g/L。
实施例5:添加剂具体包括:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.04g/L、尿苷0.04g/L、鸟苷0.04g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
实施例6:添加剂具体包括:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.1g/L、丁酸钠0.16g/L、丙戊酸0.16g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.1g/L、肼苯哒嗪0.1g/L。
实施例7:添加剂具体包括:L-天冬氨酸6.6g/L、L-天冬酰胺6.6g/L、L-精氨酸4.3g/L、L-组氨酸5.0g/L、L-异亮氨酸2.5g/L、L-亮氨酸2.5g/L、L-赖氨酸6.6g/L、L-蛋氨酸4.0g/L、L-苯丙氨酸2.75g/L、L-苏氨酸10g/L、L-色氨酸1.4g/L、L-酪氨酸7.5g/L、L-缬氨酸6.6g/L、L-半胱氨酸2.2g/L、L-谷氨酸2.74g/L、L-谷氨酰胺3.1g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
实施例8:添加剂具体包括:L-天冬氨酸6.6g/L、L-天冬酰胺6.6g/L、L-精氨酸4.3g/L、L-组氨酸5.0g/L、L-异亮氨酸2.5g/L、L-亮氨酸2.5g/L、L-赖氨酸6.6g/L、L-蛋氨酸4.0g/L、L-苯丙氨酸2.75g/L、L-苏氨酸10g/L、L-色氨酸1.4g/L、L-酪氨酸7.5g/L、L-缬氨酸6.6g/L、L-半胱氨酸2.2g/L、L-谷氨酸2.74g/L、L-谷氨酰胺3.1g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
阳离子聚合物转染方法,包括以下步骤:
Expi CHO基础培养基作为对照组,在DMEM/F-12商业化培养基基础上添加了实施例1~8的添加剂的作为实验组;
1)准备Expi CHO-S细胞,在Expi CHO基础培养基中培养,以细胞密度0.3×106细胞数/mL进行传代培养,每三天传代一次,传代三次后,转染实验备用;
2)转染实验前一天,细胞密度控制在3×106细胞数/mL~4×106细胞数/mL,实验当天,细胞计数备用;
3)用无血清培养基DMEM稀释所需的质粒和转染试剂,转染试剂室温孵育5分钟后,将稀释后的质粒和稀释后的转染试剂混合孵育10分钟,混匀时注意动作轻柔;
4)将实验用细胞,制备成细胞悬液,以6×106细胞数/mL细胞密度接种于125mL摇瓶中;
5)将质粒和转染试剂的混合液,逐滴缓慢加入至含细胞的培养基中,培养体系共20mL,放入二氧化碳摇床,进行培养(培养条件:37℃、8%二氧化碳、100-120rpm);
6)培养24小时后,二氧化碳培养箱温度由37℃降至32℃,继续培养;
7)在D01、D03、D05、D07、D09、D11、D13、D14进行细胞数量和活率的监测,并在细胞活率大于60%前,分别添加5%的Expi CHO基础培养基补料和5%上述在DMEM/F-12商业化培养基基础上添加了实施例1~8的添加剂的培养基补料;
8)分别在24小时、48小时,用流式法,检测转染效率;
9)在细胞活率降为60%以下时,取细胞上清,用高效液相色谱法,检测蛋白表达量。
如图1的活细胞密度与如图2的细胞活率的结果来看,实施例1、实施例4、实施例5、实施例6优于对照组,能够维持更高的细胞活率。
通过高效液相色谱法,分析以上优于对照组的实验组,图3为蛋白的表达量,实施例1优于其他实验组,明显高于对照组。同时为了验证添加该组合物对转染效率的影响,分别在转染24小时、48小时,通过流式检测其转染效率,图4为对照组在24小时的转染效率为67.2%,图5为添加实施例1中上述组合物在24小时的转染效率为66%,图6为对照组在48小时的转染效率为87%,图7为添加实施例1中上述组合物在48小时的转染效率为88.9%,说明添加组分不会影响其转染效率。
本发明提出了一种增强CHO细胞瞬时转染表达蛋白的培养基及其应用,搭配市售CHO瞬转基础培养基DMEM/F12培养基,通过添加组合物,补料的方式,能在细胞转染后,维持高活率,使培养周期延长,从而提高蛋白的表达量。本发明含有多种高浓度氨基酸和一些其他核苷酸及特殊成分,其中白藜芦醇、丁酸钠、丙戊酸、诺考达唑、普鲁卡因胺、肼苯哒嗪的联合作用,可显著提高蛋白产量,且不影响细胞的正常培养,高浓度氨基酸的添加,尤其L-半胱氨酸和酪氨酸的添加,可延长细胞存活的时间,从而使蛋白表达进一步提高,长达13-14天的培养,使蛋白表达提高1.4-2.24倍。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程方法物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程方法物品或者设备所固有的要素。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。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之内。
Claims (4)
1.一种CHO细胞培养基,包括基础培养基和添加剂,其特征在于,所述的基础培养基为DMEM/F12培养基,所述的添加剂为:L-天冬氨酸13.2g/L、L-天冬酰胺13.2g/L、L-精氨酸8.6g/L、L-组氨酸10g/L、L-异亮氨酸5g/L、L-亮氨酸5g/L、L-赖氨酸13.2g/L、L-蛋氨酸8g/L、L-苯丙氨酸5.5g/L、L-苏氨酸20g/L、L-色氨酸2.8g/L、L-酪氨酸15g/L、L-缬氨酸13.2g/L、L-半胱氨酸4.4g/L、L-谷氨酸5.5g/L、L-谷氨酰胺6.2g/L、对氨基苯甲酸5g/L、腺苷0.02g/L、尿苷0.02g/L、鸟苷0.02g/L、甘露聚糖7g/L、白藜芦醇0.5g/L、丁酸钠0.8g/L、丙戊酸0.8g/L、硫酸葡聚糖0.1g/L、诺考达唑0.1g/L、普鲁卡因胺0.5g/L、肼苯哒嗪0.1g/L。
2.根据权利要求1所述的一种CHO细胞培养基,其特征在于,所述的CHO细胞为CHO-S细胞。
3.根据权利要求2所述的一种CHO细胞培养基,其特征在于,所述的CHO-S细胞为ExpiCHO-S细胞。
4.根据权利要求1所述的一种CHO细胞培养基的应用,其特征在于,用于Expi CHO-S细胞的培养。
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