CN114736871A - 一种腺病毒包装wayne293 lvpro动物细胞培养方法 - Google Patents
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Abstract
本发明公开了一种腺病毒包装WAYNE293LVPRO动物细胞培养方法。是将WAYNE293LVPRO细胞培养基添加物添加到WAYNE293LVPRO细胞生产腺病毒的培养系统中进行培养，所述的WAYNE293LVPRO细胞培养基添加物，其为胱氨基酸2‑6g/L、天门冬酸双肽2‑3g/L和赖氨酸三肽2‑3g/L。利用本发明的方法可以显著的提高WAYNE293LVPRO细胞的腺病毒生产能力，以满足大量表达基因治疗性病毒的需求，对于病毒药物的产业化的意义是非常重大的。
Description
技术领域：
本发明属于生物技术领域，具体涉及一种腺病毒包装WAYNE293 LVPRO动物细胞培养方法。
背景技术：
动物细胞表达的腺病毒药物销售额迅速增长，作为商业化生产腺病毒的核心技术之一的大规模动物细胞悬浮培养成为腺病毒药物发展的新瓶颈。其中之一主要技术限制就是国内没有自己的高效能商业化动物悬浮培养基，而SIGMA、Invitrogen、Hyclone和Lonza等国际知名培养基，每升高达上千元，且配方保密，不利于后续的培养工艺优化。故研制出自主品牌且低成本的无血清无动物来源培养基，对工艺优化与反应器放大，以满足大量表达基因治疗性病毒的需求，对于病毒药物的产业化的意义是非常重大的。
市场病毒药物70％以上都是由HEK203细胞宿主细胞表达。目前WAYNE293 LVPRO细胞没有培养基添加剂专门设计用于HEK293细胞的生长和悬浮培养条件下病毒的表达。现希望发明一种新型WAYNE293 LVPRO细胞培养基添加物增加腺病毒的产量。
发明内容：
本发明的目的是提供一种腺病毒包装WAYNE293 LVPRO动物细胞培养方法。
本发明的腺病毒包装WAYNE293 LVPRO动物细胞培养方法，是将WAYNE293 LVPRO细胞培养基添加物添加到WAYNE293 LVPRO细胞生产腺病毒的培养系统中进行培养，所述的WAYNE293 LVPRO细胞培养基添加物，其为胱氨基酸2-6g/L、天门冬酸双肽2-3g/L和赖氨酸三肽2-3g/L。
优选，是将WAYNE293 LVPRO细胞培养基添加物按照体积比1:6的体积比添加到WAYNE293 LVPRO细胞生产腺病毒的培养系统中进行培养。
优选，所述的提高腺病毒生产的WAYNE293 LVPRO细胞培养基添加物，其为胱氨基酸4g/L,天门冬酸双肽2.5g/L和赖氨酸三肽2.5g/L。
优选，所述的WAYNE293 LVPRO细胞培养基添加物是将各成分按其含量加入到水中，除菌制得。
利用本发明的方法可以显著的提高WAYNE293 LVPRO细胞的腺病毒生产能力，以满足大量表达基因治疗性病毒的需求，对于病毒药物的产业化的意义是非常重大的。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
1.商业化的AAV病毒载体质粒、包装质粒和辅助质粒(均购自合肥赛海生物科技有限公司)，浓度大于1ug/ul，A260/280在1.7-1.8间方可。
2.以5×105个/ml的细胞浓度接种WAYNE293 LVPRO细胞(其公开于申请号CN202110688920.7，发明名称：一种适应无血清培养基环境的WAYNE293 LVPRO细胞及其应用的申请中，其保藏信息是：人胚胎肾细胞WAYNE 293于2021年5月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101，保藏编号为：CGMCC No.22348)，37℃，120rpm摇床培养4天后，WAYNE293 LVPRO细胞浓度达到5-10×106个/ml。
3.做脂转complex，按照以下比例混合
| 试剂名称 | 试剂数量 |
| AAV病毒载体质粒(合肥赛海生物病毒包装质粒) | 5ul(1.0ug/ul) |
| 包装质粒(合肥赛海生物病毒包装质粒) | 5ul(1.0ug/ul) |
| 辅助质粒(合肥赛海生物病毒辅助质粒) | 5ul(1.0ug/ul) |
4.取3μl脂转complex，加入步骤2的3ml含WAYNE293 LVPRO的培养液中，混匀。
5.小心加入脂转complex混合液，十字形慢慢晃动3次，在37℃5％CO2条件下，孵箱中培养90分钟，完成预培养。
6.预培养完成后，加入27ml的WAYNE293 LVPRO培养液(中山康天晟合生物技术有限公司，货号A21501)，在37℃，120rpm摇床继续培养24小时。
7. 24小时后添加5mL添加物加入到培养体系中，继续培养72小时后，然后检测病毒颗粒的数量，进行估计病毒滴度。以不加5mL添加物，加入5ml水的作为对照。每个处理3个重复。所述的添加物含有胱氨基酸4g/L,天门冬酸双肽2.5g/L和赖氨酸三肽2.5g/L的水溶液，具体制备方法是将胱氨基酸,天门冬酸双肽和赖氨酸三肽按其含量加入到水中，过滤除菌。
由于病毒颗粒同时存在于包装细胞和培养上清中，可以将细胞和培养上清都收集下来以获得最好的收率。准备一个干冰乙醇浴(将乙醇倾入装有干冰的泡沫盒即可，也可用液氮替代干冰乙醇浴)和37℃水浴。将产毒的细胞连同培养基一同收集到一个15ml的离心管中。收集细胞时，将培养盘倾斜一定角度将细胞刮到培养基中，1000rpm/min，离心3分钟，分离细胞和上清，将上清另外存放，细胞用1ml PBS重悬。将细胞悬浮液在干冰乙醇浴和37℃水浴中反复转移，冻融四次。每次融解后稍加震荡。注意：每次凝固和解冻大概需要十分钟的时间。然后检测病毒颗粒的数量(方法参见：快速病毒鉴定和定量检测方法综述，KumarPankaj University of Saskatchewan,Saskatoon,Canada，美国新泽西州普林斯顿合原研究有限责任公司(Synatom Research)DOI http://dx.doi.org/10.13070/mm.cn.3.207)。
实验结果如下：添加添加物的腺病毒产量为2.5×106个病毒载体/ml，远远高于对照的0.5×106个病毒载体/ml。由此可见，添加物能够显著增加WAYNE293 LVPRO的腺病毒产量。
实施例2：
WAYNE293 LVPRO细胞培养基添加物，其为胱氨基酸2g/L、天门冬酸双肽3g/L和赖氨酸三肽2g/L的水溶液，其制备方法同实施例1。
实施例3：
WAYNE293 LVPRO细胞培养基添加物，其为胱氨基酸6g/L、天门冬酸双肽2g/L和赖氨酸三肽3g/L的水溶液，其制备方法同实施例1。
Claims (4)
1.一种腺病毒包装WAYNE293 LVPRO动物细胞培养方法，其特征在于，是将WAYNE293LVPRO细胞培养基添加物添加到WAYNE293 LVPRO细胞生产腺病毒的培养系统中进行培养，所述的WAYNE293 LVPRO细胞培养基添加物，其为胱氨基酸2-6g/L、天门冬酸双肽2-3g/L和赖氨酸三肽2-3g/L。
2.根据权利要求1所述的方法，其特征在于，将WAYNE293 LVPRO细胞培养基添加物按照体积比1:6的体积比添加到WAYNE293 LVPRO细胞生产腺病毒的培养系统中进行培养。
3.根据权利要求1或2所述的方法，其特征在于，所述的提高腺病毒生产的WAYNE293LVPRO细胞培养基添加物，其为胱氨基酸4g/L,天门冬酸双肽2.5g/L和赖氨酸三肽2.5g/L。
4.根据权利要求1所述的方法，其特征在于，所述的WAYNE293 LVPRO细胞培养基添加物是将各成分按其含量加入到水中，除菌制得。
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