CN116790664A - 敲低suv39h1表达的cho细胞系的应用、重组蛋白表达系统 - Google Patents
敲低suv39h1表达的cho细胞系的应用、重组蛋白表达系统 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，具体涉及敲低SUV39H1基因表达的CHO细胞系的应用、重组蛋白表达系统。本发明构建靶向SUV39H1基因的shRNA表达载体，利用SSR重组酶实现干扰序列DNA载体高效稳转，催化shRNA载体整合到细胞基因组，可明显提升干扰效率；敲低SUV39H1基因表达，应用于重组蛋白表达系统，能够显著提高目的蛋白表达水平，有效解决了CHO细胞表达系统在生物制药生产过程中存在的表达水平较低的问题。
Description
技术领域
本发明属于基因工程技术领域，具体涉及敲低SUV39H1表达的CHO细胞系的应用、重组蛋白表达系统。
背景技术
中国仓鼠卵巢(Chinese Hamster Ovary,CHO)细胞是重组蛋白药物生产的首选哺乳动物表达系统宿主细胞，但在长期培养过程中仍突出存在重组蛋白表达显著下降的问题，使得筛选获得稳定高效表达的CHO细胞系费时费力。因此，通过细胞系改造的基因工程手段构建高效CHO细胞表达系统具有重要意义，也有很多研究报道通过敲除影响细胞增加分化生长能力的基因，或者引入能够促进细胞增殖分化能力的外源基因的方式提高CHO细胞作为宿主细胞对重组蛋白的表达。
DNA甲基化和组蛋白甲基化修饰等表观遗传调控对基因转录活性、基因表达沉默等具有重要作用。目前关于DNA甲基化对CHO细胞转基因表达影响的研究主要集中在启动子的修饰改造上，例如在启动子区域突变胞嘧啶，使用无CpG的或合成的启动子，或将核心CpG岛元件插入启动子。另外，利用基因敲除、基因过表达或基因沉默等细胞工程技术，也能够改变与重组蛋白表达量或重组蛋白活性相关的基因表达水平。近几年也出现通过沉默与DNA甲基化相关基因的方式改造CHO细胞，比如专利CN110257340A公开了一种Dnmt3b基因缺陷型的CHO细胞系，该细胞系中缺失的DNA甲基转移酶Dnmt3b介导的表观遗传修饰与基因的表达调控关系更加密切，在没有筛选压力时，外源蛋白的表达更稳定，表达量也明显提高。
组蛋白3第九位赖氨酸三甲基化(H3K9me3)与基因抑制和异染色质形成有关，组蛋白甲基转移酶SUV39H1主要通过三甲基化修饰H3K9从而导致转录沉默，对异染色质的形成和基因表达的调控具有重要影响。但是通过敲低或者沉默SUV39H1表达构建CHO细胞系提升重组蛋白表达的技术还未见报道。
发明内容
为了克服现有技术的缺陷，本发明的目的之一在于提供靶向SUV39H1基因的shRNA表达载体在构建重组蛋白表达系统方面的应用，能够提高重组蛋白表达量。
本发明的目的之二在于敲低SUV39H1表达的CHO细胞系在作为重组蛋白表达用宿主细胞方面的应用，能够提升重组蛋白表达量。
同时，本发明的目的之三在于提供一种重组蛋白表达系统，以本发明提供的敲低SUV39H1表达的CHO细胞系为宿主细胞，提升重组蛋白表达量。
为了实现上述发明目的，本发明采用的技术方案如下：
靶向SUV39H1基因的shRNA表达载体在构建重组蛋白表达系统方面的应用。
敲低SUV39H1基因表达的CHO细胞系作为重组蛋白表达用宿主细胞方面的应用。
可选的，所述重组蛋白选自阿达木单抗体、人血清白蛋白。
一种重组蛋白表达系统，由CHO细胞系宿主细胞、靶向SUV39H1基因的shRNA表达载体、包含重组蛋白编码基因的表达载体构建而成。
可选的，还包括SSR重组酶(Site-specific Recombinase)；所述靶向SUV39H1基因的shRNA表达载体中包含SSR元件。
可选的，其构建方法包括首先构建敲低SUV39H1表达的CHO细胞系：在重组酶作用下，将shRNA表达载体共转染至CHO细胞系宿主细胞，获得敲低SUV39H1表达的宿主细胞；
然后将包含重组蛋白编码基因的表达载体转染至敲低SUV39H1表达的宿主细胞。
可选的，还可以采用其他构建方法，包括首先将包含重组蛋白编码基因的表达载体转染至CHO细胞系，获得重组蛋白低表达细胞系；然后在重组酶作用下，将靶向SUV39H1基因的shRNA表达载体共转染至重组蛋白低表达细胞系。
可选的，还包括首先构建shRNA表达载体：根据SUV39H1的序列，设计shRNA，将shRNA核酸片段连接插入具有SSR元件的出发载体。
可选的，所述靶向SUV39H1基因的shRNA的序列为：5’-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3’。
可选的，所述出发载体为piggy BAC载体。
具体的，所述编码shRNA的核酸片段插入U6启动子下游。
本发明有益效果：
RNAi技术可以高度特异性和选择性地敲除目的基因的表达，通过载体转染将shRNA引入哺乳动物细胞，可以稳定整合shRNA并长期敲除靶基因，从而影响重组蛋白的转录与蛋白表达水平，本发明采用RNAi技术实现SUV39H1基因敲低，沉默SUV39H1基因表达，具有以下有益效果：
1)本发明针对中国仓鼠SUV39H1基因序列设计合成shRNA(5’-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3’)，利用SSR重组酶实现干扰序列DNA载体高效稳转，催化shRNA载体整合到细胞基因组，可明显提升干扰效率，实现靶基因SUV39H1干扰稳转细胞株的构建；
2)本发明通过SSR基因重组技术能够显著降低CHO细胞中SUV39H1 mRNA和蛋白表达水平，对靶基因的敲低作用显著；
3)本发明构建的SUV39H1稳定敲低细胞株具有与正常细胞无差别的生长能力，作为重组蛋白表达系统的宿主细胞，能够显著提高目的蛋白表达水平，有效解决了CHO细胞表达系统在生物制药生产过程中存在的表达水平较低的问题；
4)同时，本发明实施例证明将靶向SUV39H1基因的shRNA表达载体通过转染至重组蛋白表达系统，能够提高重组蛋白表达量。
附图说明
图1为qPCR法检测SUV39H1稳定敲低细胞株和对照组细胞的SUV39H1基因mRNA表达结果图；
图2为Digital Western(Jess)法检测SUV39H1稳定敲低细胞株和对照组细胞的SUV39H1基因蛋白表达结果图；
图3为ELISA法检测表达阿达木单抗的SUV39H1稳定敲低细胞株和对照组细胞中重组阿达木单抗的蛋白表达结果图；
图4为Digital Western(Jess)法检测表达人血清白蛋白的SUV39H1稳定敲低细胞株和对照组细胞的SUV39H1基因蛋白表达结果图；
图5为ELISA法检测表达人血清白蛋白的SUV39H1稳定敲低细胞株和对照组细胞中人血清白蛋白的蛋白表达结果图。
具体实施方式
下面结合具体实施方式对本发明作进一步描述，但本发明的保护范围并不仅限于此；
实施例1构建靶向SUV39H1基因的shRNA表达载体
本实施靶向SUV39H1基因的shRNA表达载体，其构建方法为：
1)按照NCBI GenBank中国仓鼠SUV39H1基因序列(GeneID:100764568)，根据shRNA的设计原则，设计shRNA编码核酸片段序列为5’-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3’；
2)以含有SSR元件的piggy BAC载体(购自海星生物科技有限公司)为出发载体，通过酶切、连接的方式将步骤1)合成的核酸片段插入出发载体的U6启动子的下游，构建形成含有SSR元件的shRNA转座子载体，即为靶向SUV39H1基因的shRNA表达载体。
实施例2构建敲低SUV39H1表达的CHO细胞系
本实施例提供敲低SUV39H1表达的CHO细胞系，其构建过程的具体步骤为：
1)细胞培养准备：将CHO-S细胞于37℃、5％ CO2的恒温恒湿培养箱中使用DMEM/F12培养基(含10％胎牛血清)培养。将复苏细胞传代3次以上并于转染前一天更换细胞培养基，转染前一天胰蛋白酶消化细胞铺板24孔板中的2个孔，当细胞汇合度在60％左右处于最佳增殖状态时作为转染用细胞，转染前换液为无双抗低血清培养基；
2)细胞转染：每孔加入50μL无双抗低血清培养基、2.5μg的载体和SSR重组酶，加入50μL Opti-MEM培养基，并将2μL lipo2000加入到Opti-MEM培养基中轻柔混匀。将上述24孔板中接种培养细胞的培养基弃去，并将孵育后的质粒DNA和脂质体混合液混匀后，室温放置5min。轻柔地来回晃动培养皿使混合物均匀分布，并做好标记以及记录时间。最后将包含转染复合物和细胞的培养板放在恒温培养箱(37℃，5％ CO2)中培养过夜，之后换成DMEM/F12完全培养基。转染48h后，使用Puromycin筛选；细胞汇合度达到80％后，按照1：2进行传代，药物筛选7-14天。
3)实验分为：shRNA1+SSR重组酶干扰组(R1组)及空白对照组(control)：其中shRNA1+SSR重组酶干扰组采用实施例1提供的载体按照上述步骤细胞转染得到SUV39H1稳定敲低细胞株(R1组)；空白对照组为不添加载体的空白转染，得到空白对照细胞株(control)。
实施例3有效shRNA干扰效率检测
1)qPCR实验
设计并合成靶基因SUV39H1及内参基因GAPDH进行qPCR检测的引物，如表1所示：
表1DNA引物序列表
收集SUV39H1稳定敲除细胞系(R1)及对照组(control)，Trizol裂解提取mRNA逆转录为cDNA，使用SUV39H1引物对R1及control的SUV39H1基因的mRNA表达情况进行检测。反应体系为20μL，反应条件为：95℃5min；95℃10s，60℃30s，40个循环，标准化内参为GAPDH，2-ΔΔCt法计算目的基因的相对表达量。如图1所示，结果表明，shRNA1+SSR重组酶干扰组R1中SUV39H1基因的mRNA表达量降低至control的26％。
2)Digital Western实验
Jess全自动蛋白表达分析系统(ProteinSimple公司)检测SUV39H1蛋白表达。
准备样品及抗体：将各组细胞裂解后提取的蛋白保存在裂解液中，通过BCA试剂盒测定蛋白质浓度。
准备Jess仪器：首先打开Jess连接电脑，Self-Test进行Jess硬件自检。
配置试剂：取出试剂盒PS-ST01-EZ-8和DM-001，取出DTT粉末加入40μL去离子水配制为DTT溶液；取出Master Mix粉末加入20μLDTT溶液和20μL10×Sample Buffer配制5×Master Mix(即Loading Buffer)；取出Ladder粉末加入20μL离子水配制为Ladder(即marker，分子量标准品)。
加入样品：每孔加入3μL样品(与Master Mix配置的样品)，根据加入样品终浓度及孔数计算出所需样品总体积，将配制好的样品放在95℃水浴锅中变性5min。一抗(10μL/孔)和二抗(10μL/孔)置于冰上备用。吸取Lumino-S和Peroxide各200μL配制成发光液置于冰上避光待用。
最后根据步骤说明使用反相吸液法将配制好的试剂依次加入板内。配平后室温下2500rpm离心5min；最后上机操作，取出毛细管和板子放入Jess，点击Start开始运行。
如图2所示，Jess结果表明，转染shRNA干扰载体可使稳定敲低细胞株中SUV39H1蛋白的表达显著降低。
实施例4构建敲低SUV39H1基因表达的CHO细胞增殖检测
通过细胞生物学特性检测验证SUV39H1敲低CHO细胞系能否进行正常生长和传代培养。
首先，显微镜下观察细胞形态是否正常，采用CCK-8法(Cell CountingKit-8试剂盒，碧云天Beyotime生物公司)检测细胞增殖：将R1组与control组细胞接种于96孔板中，分别于24、48和72h用酶标仪在450nm波长处测量吸光度，绘制生长曲线。
实验结果表明，敲低SUV39H1基因表达的CHO细胞能够正常进行生长增殖。
实施例5重组蛋白表达
1、重组阿达木单抗(Adalimumab,ADM)
1)构建表达阿达木的SUV39H1稳定敲低细胞系及细胞生物学特性检测
为检测SUV39H1稳定敲低CHO细胞系对重组蛋白(抗体)表达的作用，利用表达重组阿达木单抗真核表达载体，分别转染SUV39H1稳定敲除细胞(实施例2的R1组)和CHO-S细胞(实施例2的control组)。转染后的细胞池使用杀稻瘟菌素稳定筛选14天，构建表达ADM的SUV39H1稳定敲低细胞(ADM-R1)，对照细胞(control)。
对于贴壁培养ADM-R1细胞、control细胞，CCK-8法检测细胞增殖；对于悬浮培养细胞，将重组CHO细胞(ADM-R1和control)在六孔板中以3×105cell/mL接种于化学成分确定的CD CHO培养基(河南普诺易生物制品研究院有限公司)中悬浮培养培养7天，每天收集细胞使用台盼蓝染色法通过BioTech细胞计数仪(上海睿钰生物科技有限公司)检测细胞活率和活细胞密度。实验结果表明，ADM-R1细胞生长状态、形态、细胞增殖、活细胞密度等生物学特性与正常CHO细胞无显著性差别。
2)ELISA检测重组ADM表达水平
将化学成分确定的CD CHO中悬浮培养培养7天的ADM-R1细胞、control细胞于第7天离心收集上清液，ELISA法检测重组ADM表达。如图3所示，结果表明，与control细胞组相比，SUV39H1稳定敲低细胞株重组ADM的表达水平提高了1.45倍。
2、人血清白蛋白(Human Serum Albumin，HSA)
1)构建表达人血清白蛋白的SUV39H1稳定敲低细胞及细胞生物学特性检测
本发明利用表达重组蛋白人血清白蛋白真核载体转染CHO-S细胞，转染后的细胞池使用杀稻瘟菌素稳筛14天，获得表达HSA的CHO细胞池，使用有限稀释法获得HSA低表达单克隆细胞株(control)；
将实施例1构建的靶向SUV39H1基因的shRNA表达载体在SSR重组酶作用下转染至HSA低表达单克隆中，使用Puromycin筛选获得表达HSA的SUV39H1稳定敲低细胞株(HSA-R1)，如图4所示，其中control组为HSA低表达单克隆细胞株；
CCK-8法检测贴壁培养HSA-R1细胞增殖；对于悬浮培养细胞，将HSA-R1细胞、HSA低表达单克隆细胞(control)在六孔板中以3×105cell/mL接种于CD CHO培养基中，台盼蓝染色法检测活细胞密度和细胞活率。实验结果表明，HSA-R1细胞增殖与对照组细胞无显著性差别。
2)ELISA检测HSA表达变化
悬浮培养第7天低温离心收集上清液，ELISA法检测表达HSA的SUV39H1稳定敲低细胞株HSA-R1与对照组细胞HSA低表达单克隆细胞株中重组蛋白表达变化。如图5所示，结果表明，与control细胞组相比，SUV39H1稳定敲低细胞株HSA的表达水平提高了2.63倍。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：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；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.靶向SUV39H1基因的shRNA表达载体在构建重组蛋白表达系统方面的应用。
2.敲低SUV39H1表达的CHO细胞系作为重组蛋白表达用宿主细胞方面的应用。
3.如权利要求1所述的应用，其特征在于，所述重组蛋白选自阿达木单抗体、人血清白蛋白。
4.一种重组蛋白表达系统，其特征在于，由CHO细胞系宿主细胞、靶向SUV39H1基因的shRNA表达载体、包含重组蛋白编码基因的表达载体构建而成。
5.如权利要求4所述的重组蛋白表达系统，其特征在于，还包括SSR重组酶；所述靶向SUV39H1基因的shRNA表达载体中包含SSR元件。
6.如权利要求5所述的重组蛋白表达系统，其特征在于，其构建方法包括首先构建敲低SUV39H1表达的CHO细胞系：在重组酶作用下，将靶向SUV39H1基因的shRNA表达载体共转染至CHO细胞系宿主细胞，获得敲低SUV39H1表达的宿主细胞；
然后将包含重组蛋白编码基因的表达载体转染至敲低SUV39H1表达的宿主细胞。
7.如权利要求5所述的重组蛋白表达系统，其特征在于，其构建方法包括首先将包含重组蛋白编码基因的表达载体转染至CHO细胞系，获得重组蛋白低表达细胞系；然后在重组酶作用下，将靶向SUV39H1基因的shRNA表达载体共转染至重组蛋白低表达细胞系。
8.如权利要求6或7所述的重组蛋白表达系统，其特征在于，还包括首先构建shRNA表达载体：根据SUV39H1的序列，设计shRNA，将shRNA核酸片段连接插入具有SSR元件的出发载体。
9.如权利要求6所述的重组蛋白表达系统，其特征在于，所述靶向SUV39H1基因的shRNA的序列为：
5’-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3’。
10.如权利要求7所述的重组蛋白表达系统，其特征在于，所述出发载体为piggy BAC载体；所述编码shRNA的核酸片段插入U6启动子下游。
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