CN113061584A - 一种能促进腺病毒生长的培养基添加物 - Google Patents
一种能促进腺病毒生长的培养基添加物 Download PDFInfo
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Abstract
本发明公开了一种能促进腺病毒生长培养基添加物，属于细胞培养技术领域。包括：氯化钠，莫能菌素，磷酸氢二钠，葡萄糖,谷氨酰胺，盐酸腐胺，柠檬酸铁，乙二醇，磷脂，胆固醇，核糖核酸，白蛋白。将促病毒生长添加物加入到腺病毒培养基，可使腺病毒产量增加，为腺病毒生长提供基础。
Description
技术领域
本发明属于细胞培养技术领域，具体涉及一种能促进腺病毒生长的培养基添加物。
背景技术
随着分子生物学的迅速发展，在实验室构建各种载体、克隆及分析目标基因，使疾病深入至分子水平研究成为可能，基因诊断及基因治疗技术也随之诞生。
目前基因治疗研究的热门方法是将外源基因DNA或RNA片段导入靶细胞或组织，研究靶基因的上调或抑制情况，从而而选择合适高效的基因导入介质系统尤为关键，而病毒载体也随之成为了当前基因治疗载体研究的热点，其优势在于：
1、病毒包装技术经历了多年的研究，已趋于成熟，可用于产业化大量生产；
2、病毒基因组的结构简单、分子背景比较清楚，稳定易于改造、易于制备；
3、病毒的宿主范围广，且具有高效的靶向特异性；
4、在目的基因表达方面相对于脂质体介导的效果更明显、长时、稳定；
5、通过载体改造的方式形成了复制缺陷型结构，安全性高；
6、可提高RNA干扰和基因过表达效率，增加实验成功率。
病毒介导是借助于病毒颗粒作为基因的载体，利用病毒的侵染特性，自发的侵染哺乳动物细胞，将目的基因导入哺乳动物细胞中，以完成目的产物的超量表达或干涉。该技术目前在基因、小RNA、转录因子等功能验证领域以及重组蛋白表达、转基因哺乳动物制备等领域作为一种主要的技术手段被广泛使用。该技术体系主要由病毒包装和病毒侵染筛选两个部分组成。病毒包装就是借助基因工程的方法将目的基因片段与人为改造病毒的基因组通过特殊的位点连接起来形成重组病毒基因组以及重组病毒制备的过程，常规使用的病毒包装系统有慢病毒包装系统和腺病毒包装系统。包装好的慢病毒和腺病毒都可以自主的侵染哺乳动物细胞，导入外源基因。
用于病毒包装的载体主要有腺病毒载体和慢病毒载体。
腺病毒感染效率高，很多细胞都能达到近99％，纯化后的腺病毒可以直接进行动物活体注射，可以在体外扩增，每次使用完后，可以自己用包装细胞进行扩增，节约成本。
但也存在较多问题，腺病毒包装周期比慢病毒较长(一般要两个半月到三个月)，产量低，病毒增殖不稳定，商业化配方保密，不明确等因素，导致腺病毒培养不稳定。
因此，如何提供一种能促进腺病毒生长的培养基添加物是本领域亟待解决的问题。
发明内容
本发明公开了一种能促进腺病毒生长的培养基添加物。
为了实现上述目的，本发明采用如下技术方案：
一种能促进腺病毒生长培养基添加物，包括：氯化钠、莫能菌素、磷酸氢二钠、葡萄糖、谷氨酰胺、盐酸腐胺、柠檬酸铁、乙二醇、磷脂、胆固醇、核糖核酸和白蛋白；
一种能促进腺病毒生长培养基添加物，包括：氯化钠1-5g/L、莫能菌素50-90mg/L、磷酸氢二钠100-200mg/L、葡萄糖1-5g/L、谷氨酰胺0.1-0.5g/L、盐酸腐胺1-5mg/L、柠檬酸铁100-400mg/L、乙二醇1％-5％体积比、磷脂1-20mg/L、胆固醇1-25mg/L、核糖核酸1-50μg/L和白蛋白1-5g/L；
一种能促进腺病毒生长培养基添加物，包括：氯化钠3g/L、莫能菌素83mg/L、磷酸氢二钠140mg/L、葡萄糖1g/L、谷氨酰胺0.15g/L、盐酸腐胺2mg/L、柠檬酸铁400mg/L、乙二醇1％体积比、磷脂10mg/L、胆固醇5mg/L、核糖核酸20μg/L和白蛋白2g/L；
一种利用促进腺病毒生长培养基添加物的培养方法，包括以下步骤：
(1)活化培养宿主细胞；
(2)将腺病毒和活化后的宿主细胞加入至10倍浓度的上述促进腺病毒生长的培养基添加物中，混匀，通入CO2，37℃条件下预培养90分钟；
(3)预培养后，加入腺病毒培养基，使促进腺病毒生长培养基添加物稀释为1倍浓度，继续培养至完成；
优选的，步骤(1)中，所述活化培养宿主细胞的条件为：37℃，120rpm；
优选的，步骤(1)中，宿主细胞种类包括：CHO细胞和293细胞；
优选的，步骤(2)中，CO2浓度为5％。
优选的，步骤(3)中，所述腺病毒培养基为Expi293TM培养基；
优选的，步骤(3)中，继续培养方式为悬浮培养；
优选的，宿主细胞以5×105个/ml的细胞浓度接种，达到5×106个/ml细胞浓度时停止；
优选的，活化培养基为Expi293TM培养基；
优选的，步骤(2)中，腺病毒MOI值为5；
优选的，步骤(3)中，继续培养条件为：37℃，120rpm；
综上所述，本发明公开了一种能促进腺病毒生长的培养基添加物，包括：氯化钠，莫能菌素，磷酸氢二钠，葡萄糖,谷氨酰胺，盐酸腐胺，柠檬酸铁，乙二醇，磷脂，胆固醇，核糖核酸，白蛋白，将促病毒生长添加物加入到腺病毒培养基，可使腺病毒产量增加，为腺病毒生长提供基础。
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。
促进腺病毒生长培养基添加物1
配制10X的浓储液：取10倍量的氯化钠1g/L、莫能菌素50mg/L、磷酸氢二钠100mg/L、葡萄糖1g/L、谷氨酰胺0.1g/L、盐酸腐胺1mg/L、柠檬酸铁100mg/L、乙二醇1％体积比、磷脂1mg/L、胆固醇1mg/L、核糖核酸1μg/L和白蛋白1g/L，溶于1L的DMEM培养基中；备用。
促进腺病毒生长培养基添加物2
配制10X的浓储液：取10倍量的氯化钠5g/L、莫能菌素90mg/L、磷酸氢二钠1200mg/L、葡萄糖5g/L、谷氨酰胺0.5g/L、盐酸腐胺5mg/L、柠檬酸铁400mg/L、乙二醇5％体积比、磷脂20mg/L、胆固醇25mg/L、核糖核酸50μg/L和白蛋白5g/L，溶于1L的PBS缓冲液中；备用
促进腺病毒生长培养基添加物3
配制10X的浓储液：取10倍量的氯化钠3g/L，莫能菌素83mg/L，磷酸氢二钠140mg/L，葡萄糖1g/L，谷氨酰胺0.15g/L，盐酸腐胺2mg/L，柠檬酸铁400mg/L，乙二醇1％体积比，磷脂10mg/L，胆固醇5mg/L，核糖核酸20μg/L，白蛋白2g/L，溶于1L的蒸馏水中；备用
实施例1
1.在125ml的平底锥形瓶中加入20ml Expi293TM培养液，以5×105个/ml的细胞浓度接种293细胞，37℃，120rpm摇床培养4天后，293细胞浓度达到5×106个/ml。
2.取3μl首次扩增的腺病毒保存液，加入促进腺病毒生长培养基添加物2至3ml，混匀，将病毒稀释得到的MOI值约为5，得病毒混合液。
3.移去Expi293TM培养液，小心加入病毒混合液，切勿破坏细胞单层，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。
4.预培养完成后，加入27ml的Expi293TM培养液，在37℃，120rpm摇床继续培养72小时，检测病毒颗粒的数量，进行MOI测定以估计病毒颗粒，结果如表1所示。若如果此时得到的病毒量已足够，可立即进行病毒滴度测定。收集细胞，600×g离心5分钟沉淀细胞，去上清后加入最小体积(一般为原始体积的1/10即1ml)病毒保存溶液重悬细胞。-200C/370C冻融3次，台式离心机上以最大速率离心去除细胞碎片，收集上清，然后进行病毒滴定，结果如表1所示。
实施例2
1.在125ml的平底锥形瓶中加入20ml Expi293TM培养液，以5×105个/ml的细胞浓度接种293细胞，37℃，120rpm摇床培养4天后，293细胞浓度达到5×106个/ml。
2.取3μl首次扩增的腺病毒保存液，加入促进腺病毒生长培养基添加物1至3ml，混匀，将病毒稀释得到的MOI值约为5，得病毒混合液。
3.移去Expi293TM培养液，小心加入病毒混合液，切勿破坏细胞单层，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。
4.预培养完成后，加入27ml的Expi293TM培养液，在37℃，120rpm摇床继续培养72小时，检测病毒颗粒的数量，进行MOI测定以估计病毒颗粒，结果如表1所示。若如果此时得到的病毒量已足够，可立即进行病毒滴度测定。收集细胞，600×g离心5分钟沉淀细胞，去上清后加入最小体积(一般为原始体积的1/10即1ml)病毒保存溶液重悬细胞。-200C/370C冻融3次，台式离心机上以最大速率离心去除细胞碎片，收集上清，然后进行病毒滴定，结果如表1所示。
实施例3
1.在125ml的平底锥形瓶中加入20ml Expi293TM培养液，以5×105个/ml的细胞浓度接种CHO细胞，37℃，120rpm摇床培养4天后，CHO细胞浓度达到5×106个/ml。
2.取3μl首次扩增的腺病毒保存液，加入促进腺病毒生长培养基添加物3至3ml，混匀，将病毒稀释得到的MOI值约为5，得病毒混合液。
3.移去Expi293TM培养液，小心加入病毒混合液，切勿破坏细胞单层，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。
4.预培养完成后，加入27ml的Expi293TM培养液，在37℃，120rpm摇床继续培养72小时，检测病毒颗粒的数量，进行MOI测定以估计病毒颗粒，结果如表1所示。若如果此时得到的病毒量已足够，可立即进行病毒滴度测定。收集细胞，600×g离心5分钟沉淀细胞，去上清后加入最小体积(一般为原始体积的1/10即1ml)病毒保存溶液重悬细胞。-200C/370C冻融3次，台式离心机上以最大速率离心去除细胞碎片，收集上清，然后进行病毒滴定。
对比例1
除步骤2中将促进腺病毒生长培养基添加物1换成DMEM培养基外，其余操作与实施例1相同。
对比例2
除步骤2中将促进腺病毒生长培养基添加物2换成PBS缓冲液外，其余操作与实施例2相同。
对比例3
除步骤2中将促进腺病毒生长培养基添加物3换成蒸馏水外，其余操作与实施例3相同。
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 (10)
1.一种能促进腺病毒生长培养基添加物，其特征在于，包括：氯化钠、莫能菌素、磷酸氢二钠、葡萄糖、谷氨酰胺、盐酸腐胺、柠檬酸铁、乙二醇、磷脂、胆固醇、核糖核酸和白蛋白。
2.一种能促进腺病毒生长培养基添加物，其特征在于，包括：氯化钠1-5g/L、莫能菌素50-90mg/L、磷酸氢二钠100-200mg/L、葡萄糖1-5g/L、谷氨酰胺0.1-0.5g/L、盐酸腐胺1-5mg/L、柠檬酸铁100-400mg/L、乙二醇1％-5％体积比、磷脂1-20mg/L、胆固醇1-25mg/L、核糖核酸1-50μg/L和白蛋白1-5g/L。
3.一种能促进腺病毒生长培养基添加物，其特征在于，包括：氯化钠3g/L、莫能菌素83mg/L、磷酸氢二钠140mg/L、葡萄糖1g/L、谷氨酰胺0.15g/L、盐酸腐胺2mg/L、柠檬酸铁400mg/L、乙二醇1％体积比、磷脂10mg/L、胆固醇5mg/L、核糖核酸20μg/L和白蛋白2g/L。
4.一种利用促进腺病毒生长培养基添加物的培养方法，其特征在于，包括以下步骤：
(1)活化培养宿主细胞；
(2)将腺病毒和活化后的宿主细胞加入至10倍浓度的如权利要求1-3任一所述的促进腺病毒生长的培养基添加物中，混匀，通入CO2，37℃条件下预培养90分钟；
(3)预培养后，加入腺病毒培养基，使促进腺病毒生长培养基添加物稀释为1倍浓度，继续培养至完成。
5.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(1)中，所述活化培养宿主细胞的条件为：37℃，120rpm。
6.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(1)中，所述宿主细胞种类为：CHO细胞或293细胞。
7.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(2)中，腺病毒MOI值为5。
8.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(3)中，继续培养条件为：37℃，120rpm。
9.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(3)中，腺病毒培养基为Expi293TM培养基。
10.如权利要求4所述的促进腺病毒生长的培养方法，其特征在于，步骤(3)中，所述继续培养方式为悬浮培养。
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Inventor after: Zhao Junling Inventor after: Huang Xinpeng Inventor after: Wang Jing Inventor after: Chen Yan Inventor after: Swiftlet Inventor after: Wang Ruiyao Inventor before: Zhao Junling Inventor before: Huang Xinming Inventor before: Wang Jing Inventor before: Chen Yan Inventor before: Swiftlet Inventor before: Wang Ruiyao |
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Effective date of registration: 20211122 Address after: 010000 No.4, area B, East Branch Road, Yulong Industrial Park, Yuquan District, Hohhot City, Inner Mongolia Autonomous Region Applicant after: INNER MONGOLIA JIN YUAN KANG BIOTECHNOLOGY CO.,LTD. Address before: 010000 No.4, area B, East Branch Road, Yulong Industrial Park, Yuquan District, Hohhot City, Inner Mongolia Autonomous Region Applicant before: Zhao Junling |
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Address after: 010000 No.4, area B, East Branch Road, Yulong Industrial Park, Yuquan District, Hohhot City, Inner Mongolia Autonomous Region Applicant after: Inner Mongolia jinyuankang Bioengineering Co.,Ltd. Address before: 010000 No.4, area B, East Branch Road, Yulong Industrial Park, Yuquan District, Hohhot City, Inner Mongolia Autonomous Region Applicant before: INNER MONGOLIA JIN YUAN KANG BIOTECHNOLOGY CO.,LTD. |
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Application publication date: 20210702 |
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