CN116716253A - Gs基因敲除的cho细胞株及其构建方法与应用 - Google Patents
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Abstract
本申请属于生物技术领域,公开了一种GS基因敲除的CHO细胞株及其构建方法与应用。本申请GS基因敲除的CHO细胞株构建方法,采用的sgRNA截短成17bp,大大提高了敲除效率,降低了脱靶率。本申请所述的一株内源性GS基因敲除的悬浮CHO‑K1细胞株,可以减少MSX的使用,并且能够达到更严格的GS筛选条件,作为宿主细胞系,其筛选周期大大缩短,同时可以提高细胞的生产力,提高重组蛋白的表达量,在代谢方面减少代谢废物乳酸的生成。
Description
技术领域
本申请涉及生物技术领域,特别涉及一种GS基因敲除的CHO细胞株及其构建方法与应用。
背景技术
在临床试验中,越来越多的生物制药候选药物迫切需要简化细胞系开发过程。迄今为止,基于中国仓鼠卵巢细胞(CHO)的表达系统仍然是最常见的哺乳动物细胞表达系统,可以用于生产具有复杂糖型的重组蛋白。其优势在于其对无蛋白培养基的悬浮培养体系的适应性,增殖迅速,易于遗传操作,糖基化谱与人类相似,可以满足大规模悬浮培养。
在CHO细胞表达系统中,二氢叶酸还原酶(DHFR)和谷氨酰胺合成酶(GS)选择系统是最常用的。相比较DHFR选择系统,GS选择系统提供了时间优势,因为它需要更少的基因拷贝就能使稳定的细胞存活,因此可以更快地选择高产细胞。GS选择系统利用GS的基本活性催化谷氨酸和氨的ATP依赖的缩合生成谷氨酰胺,需要使用甲硫氨酸硫氧胺(MSX)和不含谷氨酰胺的培养基以允许使用GS作为显性选择标记。然而,为了缩短和改进细胞系开发过程,可以通过创建GS敲除(GS-KO)宿主来提高细胞系构建开发过程的性能,与CHO-K1细胞相比,GS-KO细胞允许更严格的GS选择条件,因此可以显著提高批量细胞培养的生产力,同时存活的非生产者克隆更少。
因此,如何获得GS基因敲除的CHO-K1细胞株成为本领域内的重要研究课题。目前,可以通过基因组编辑技术来敲除CHO-K1细胞里的GS基因,构建GS-KO细胞株,基因编辑技术有:ZEN:锌指核酸酶;TALENs:转录激活因子样效应物核酸酶和CRISPR-cas9等。
其中,细菌适应性免疫系统CRISPR(成簇的规律间隔回文重复序列)-Cas(CRISPR相关蛋白)已被广泛用于哺乳动物基因组的序列特异性编辑。Cas9核酸酶蛋白与20个核苷酸(nt)序列的引导RNA(sgRNA)一起作用,用于引入位点特异性双链断裂。Cas9-sgRNA复合物对DNA的靶向是通过sgRNA和DNA之间的碱基配对以及相邻NGG PAM(前间隔序列邻近基序)序列的存在来确定的。双链断裂发生在PAM位点上游3bp,允许通过替代DNA修复途径进行靶向序列修饰:非同源末端连接(NHEJ)引入移码插入和缺失突变导致功能等位基因丢失或同源定向修复(HDR)用于在目标位点精确插入点突变或所需序列的片段。然而,CRISPR/Cas9的脱靶效应已经广为人知。
发明内容
发明目的:针对上述现有技术中存在的技术问题,本申请提供了一种大大提高敲除效率、降低脱靶率的GS基因敲除的CHO细胞株构建方法,以及构建得到的GS基因敲除的CHO-K1细胞株,以及该细胞株在重组蛋白表达中的应用。
技术方案:本申请所述的GS基因敲除的CHO细胞株构建方法,包括以下步骤:
(1)cas9 circRNA和sgRNA构建:合成含有编码cas9蛋白的序列和附属元件的cas9线性化双链DNA,通过T7 RNA聚合酶体外转录成线性化RNA,再将线性化RNA环化成circRNA;针对CHO细胞GS基因的CDS序列,设计sgRNA靶向序列,体外转录成靶向sgRNA;
(2)转染及流式分选:将cas9 circRNA与sgRNA共转染CHO细胞,电转后流式分选;将存活的细胞进行分离,获得单克隆细胞,静置培养,得到单克隆细胞株;
(3)筛选与鉴定:将同一株单克隆细胞株中的一部分置于含有谷氨酰胺的培养基中培养,将一部分置于不含有谷氨酰胺的培养基中培养;在含有谷氨酰胺的培养基中能存活,但在不含谷氨酰胺的培养基中不能存活的细胞株,即为GS基因敲除的CHO细胞株,并通过western blot进一步鉴定,无GS蛋白的细胞确定为GS基因敲除的CHO细胞株。
作为一种技术方案,步骤(1)中,所述CHO细胞GS基因的CDS编码序列如SEQ IDNO.1所示;所述sgRNA序列为:5’-ttgggctctttgcggaa-3’(SEQ ID NO.2)。
作为一种技术方案,步骤(1)中,sgRNA的构建是将sgRNA靶向序列与T7 Promoter、gRNA scaffold(76bp tracrRNA)和cPPT/CTS拼接成线性dsDNA,再体外转录成靶向sgRNA。
作为一种技术方案,步骤(1)中,所述cas9为环化cas9 circRNA。本申请使用cas9circRNA,可以提高RNA的稳定性,确保其转染进细胞内稳定发挥作用,在敲除目的序列之后发生降解,不会整合在宿主细胞基因组内。
作为一种技术方案,步骤(2)中,电转总用量为10μg-30μg,优选20μg-30μg,最优选为20μg。电转总用量为20μg是效果最好的最小用量。
作为一种技术方案,电转中cas9 circRNA:sgRNA为1:2-5;优选1:2-4;最优选为1:3。
作为一种技术方案,步骤(2)中,电转仪是Bio-rad,电转参数最优选为250V,20ms,2次,5s,4mm。也可以是其他电转参数:250V,10ms,1次,0s,4mm;250V,10ms,2次,5s,4mm;250V,20ms,1次,0s,4mm。
本申请所述的一种GS基因敲除的中国仓鼠卵巢细胞CHOK1/GSKO-C2,2023年2月22日保藏于中国典型培养物保藏中心,保藏地址为中国武汉,武汉大学,保藏编号为CCTCCNO:C202342。
所述GS基因敲除的CHO细胞株在重组蛋白表达中的应用也在本申请的保护范围内。
有益效果:相比较于现有技术,本申请GS基因敲除的CHO细胞株构建方法,采用环化的cas9 circRNA,使短半衰期的mRNA稳定,确保其转染进细胞内稳定发挥作用,在敲除目的序列之后发生降解,不会整合在宿主细胞基因组内;采用的sgRNA截短成17bp,大大提高了敲除效率,降低了脱靶率。本申请所述的一株内源性GS基因敲除的悬浮CHO-K1细胞株,可以减少MSX的使用,并且能够达到更严格的GS筛选条件,作为宿主细胞系,其筛选周期大大缩短,同时可以提高细胞的生产力,提高重组蛋白的表达量,在代谢方面减少代谢废物乳酸的生成。
附图说明
图1是GS敲除单克隆Western blot鉴定结果(M:Marker;CHO-K1细胞,是未经过改造的ATCC细胞;GS蛋白条带大小为42.3KDa);
图2是截短组GSKO克隆传代稳定性验证结果;
图3是GSKO克隆与CHO-K1细胞电转EGFP质粒筛选阶段的EGFP阳性率;
图4是常规组GSKO克隆与CHO-K1细胞表达阶段的蛋白定量;
图5是截短组GSKO克隆与CHO-K1细胞表达阶段的蛋白定量;
图6是常规组GSKO克隆与CHO-K1细胞表达阶段的乳酸水平变化;
图7是截短组GSKO克隆与CHO-K1细胞表达阶段的乳酸水平变化。
具体实施方式
下面结合具体实施例对本申请技术方案作出详细说明。
实施例1
内源性GS基因敲除的悬浮CHO-K1细胞株构建方法,包括以下步骤:
(1)cas9 circRNA/sgRNA构建
对于环化cas9 circRNA的构建,首先构建出cas9线性化dsDNA质粒。以cas9质粒(eSpCas9(BB)-2A-Puro,金斯瑞)为模板,设计引物,PCR的方式扩增出espCas9片段(使用线性化质粒PCR的试剂,Vazyme,P312),该质粒载体还包括T7 Promoter,5'Strong homologyarms,3'Strong homology arms,Ana self-splicing intron 3',Ana self-splicingintron 5',internal ribosome entry site(IRES),kozak,核定位信号序列SV40 NLS与nucleoplasmin NLS。载体两侧分别有一个40bp的5'Spacer和3'Spacer以及30bp的polyAC序列。然后,PCR产物胶回收(Vazyme,DC301)后使用T7 HighYieldRNATranscription Kit(Vazyme,TR101-01)通过体外转录将线性化质粒模板合成circRNA前体。体外转录之后,产物用DNase I(Vazyme,EN401)37℃反应30min来消化DNA模板。DNase I消化后经过核酸提取仪提纯的RNA加热到70℃反应5min,然后立即置于冰上3min。然后,将终浓度为2mM的5’-三磷酸鸟苷(GTP)以及含镁的缓冲液((50mM Tris-HCl,10mM MgCl2,1mM DTT,pH 7.5)加入反应,在55℃下孵育15min催化环状RNA循环。最后,使用核酸提取仪进行磁珠纯化(Vazyme,N412),RNase R(碧云天,R7092L)消化线性RNA片段,纯化后的circRNA使用Qubit(Thermo)测定浓度,存于-80℃。
中国仓鼠卵巢细胞CHO-K1 GS基因的CDS序列如下所示:
5’-atggccacctcagcaagttcccacttgaacaaaggcatcaagcaaatgtacatgtccctgccccagggtgagaa agtccaagccatgtatatctgggttgatggtaccggagaaggactgcgctgcaaaacccgcaccctggactgtgagcccaagtgtgtagaagagttacctgagtggaattttgatggctctagtacctttcagtctgagggctccaacagtgacatgtatctcagccctgttgccatgtttcgggaccccttccgcaaagagcccaacaagctggtgttctgtgaagtcttcaagtacaaccggaagcctgcagagaccaatttaagacacacgtgtaaacggataatggacatggtgagcaaccagcacccctggtttggaatgg aacaggagtatactctcttgggaacagatgggcacccttttggttggccttccgatggcttccctgggccccaaggtctgtattactgtggtgtgggcgcagacaaagcctatcgcagggatatcatggaggctcactaccttgcctgcttgtatgctggggtcaagattacaggaacatatgctgaggtcaagcatgcccagtgggaattccaaataggaccctgtgaaggaatccgcatgggagatcatctgtgggtggcccgtttcatcttgcatcgagtatgtaaagactttggggtaatagcaacctttgaccccaagcccattcctgggaactggaatggtgcaggctgccataccaactttagtaccaagaccatgcgggaggagaatggtctgaagcacatcaaggaggccattgagaaactaagcaagcggcaccggtaccatattcgagcctacgatcccaagggggggctggacaatgcccgtcgtctgactgggttccacaaaacgtccaacatcaacgacttttcagctggcgtcgccgatcgcagtgccagcatccgcattccccggactgtcggccaggagaagaaaggttactttgaagcccgctgcccctctgccaattgtgacccctttgcagtgacagaagccatcgtccgcacatgccttctcaatgagactggcgaccagcccttccaatacaaaaactaa-3’(SEQ ID NO.1)
针对GS基因上的CDS序列,在张锋实验室的在线预测网站,即http://crispr.tefor.net/上设计常规20bp的sgRNA靶向序列,在20bp的基础上删掉3个碱基得到截短为17bp的sgRNA靶向序列,序列分别如下:
常规sgRNA:5’-ttgttgggctctttgcggaa-3’(SEQ ID NO.3)
截短sgRNA:5’-ttgggctctttgcggaa-3’(SEQ ID NO.2)
将T7 Promoter、sgRNA靶向序列、gRNA scaffold(76bp tracrRNA)和cPPT/CTS构建成线性dsDNA,再使用T7 High Yield RNA Transcription Kit(Vazyme,TR101-01)通过体外转录将线性化质粒模板合成RNA。体外转录之后,产物用DNase I(Vazyme,EN401)37℃反应30min来消化DNA模板,消化后的产物使用核酸提取仪进行磁珠纯化(Vazyme,N412),纯化后的RNA使用Qubit(Thermo)测定浓度,存于-80℃。
(2)转染及流式分选
将来源于ATCC的贴壁CHO-K1细胞(ATCC CCL-61)驯化成悬浮细胞:复苏贴壁ATCCCHO-K1于F-12K(Hyclone,SH30526.01)+10%FBS(Gibco,10099-141c)中,用F-12K(+10%FBS)+CD CHO培养基(Gibco,10743029)进行悬浮减血清间接驯化,依次从10%FBS到5%FBS,从5%FBS到2%FBS,直至血清减为0,完全在CD CHO培养基中悬浮培养。
准备1*10^7个/mL的处于对数生长期的悬浮CHO-K1细胞,用电转方式向CHO-K1中分别转染Cas9 circRNA和sgRNA,使用的电转仪是Bio-rad,电转参数为250V,20ms,2次,5s,4mm。
电转总用量均为20μg,其中,cas9 circRNA:截短sgRNA为1:3,cas9 circRNA:常规sgRNA为1:3。电转后转移至10mL CD CHO+6mM GlutaMAX培养基中,37℃,5%CO2静置培养48h后,sony流式分选单克隆细胞于96孔板(JET,TCP-001-096)中。
(3)细胞筛选
流式分选12天之后,待细胞在96孔板中生长起来,汇合度达到50%以上,分3板进行培养,1板为保种板,所用培养基为:CHO克隆培养基(Sigma-Aldrich,C6366)+10%FBS+6mM GlutaMAX;第2板为GlutaMAX板,所用培养基为:50%CD CHO+50%CHO-K1培养上清+6mM GlutaMAX;第3板为GlutaMAX free板,所用培养基为:50%CD CHO+50%CHO-K1培养上清。GlutaMAX板和GlutaMAX free板的细胞均分,细胞数量相同。饥饿处理20天,通过台盼蓝(sigma,T6146-25G)染色判断GlutaMAX free板的死活,活率。挑选GlutaMAX free板不增殖、死亡的保种板细胞,在T12.5方瓶中悬浮培养。
(4)Westernblot
每个克隆取2.0*10^6cells,离心弃上清,加100ul RIPA裂解液(碧云天,P0013C),室温裂解10min,12000g离心5min,取上清,加25uL 5*loading,沸水浴5min变性,待western验证。
蛋白样品上样量:10ul/样品,以正常CHO-K1总蛋白为对照,每个样品需跑两块胶。电泳条件:80V,1.5h。转膜条件:恒流转膜,350mA,40min。封闭条件:5%脱脂奶粉(雀巢)(于1*PBST中配制),室温摇床孵育1h。一抗:β-actin(Vazyme,Ab101-01)1:1000稀释,GSantibody(Abcam,ab49873)1:5000稀释,抗体稀释均用5%脱脂奶粉稀释,每个样品的两块膜,一块孵育β-actin,一块孵育GS antibody,室温孵育1h或4℃过夜。洗膜:1*PBST,5min*4次。二抗孵育:GS一抗对应兔二抗(Jackson,111-005-003),1:5000;β-actin一抗对应鼠二抗(Jackson,115-005-003),1:5000。室温孵育1.5h。洗膜:1*PBST,5min*4次。最后显影。
实施例2
GS功能缺陷细胞株表型鉴定
分别电转Cas9 circRNA/常规sgRNA和Cas9 circRNA/截短sgRNA后,流式分选单个细胞。两组分别铺板10块96孔板,常规组的克隆形成率为42.8%,截短组的克隆形成率为44.2%,克隆形成率无显著差异。从中各挑选出400株生长状态较佳的克隆进行分板饥饿处理。通过不含GlutaMAX的培养基饥饿处理20天,初步筛选出培养基中不含GlutaMAX,其细胞死亡的单克隆。结果显示,截短组筛选出20株GS功能缺陷的细胞株,命名为GSKO-C1、GSKO-C2、GSKO-C3、……GSKO-C20;常规组筛选出10株GS功能缺陷的细胞株,命名为GSKO-C21、GSKO-C22、……GSKO-C30。
GS敲除单克隆Western blot鉴定
上述30株细胞均从96孔板中扩大至T12.5方瓶中悬浮培养,培养基是CD CHO+6mMGlutaMAX。待细胞密度达到对数期1.0*10^6cells/mL以上,传代扩大,第二代冻存并取2.0*10^6cells制WB样,进行Western blot鉴定GS基因是否完全敲除。结果如图1所示,截短组C1-C20均没有压出GS条带,表明GS基因已完全敲除;常规组10株中,C25可以压出GS条带,表明GS基因没有敲除干净,其余9株GS基因敲除干净。
综上所述,截短组的敲除效率明显高于常规组,是常规组的翻倍。从另一方面也可以说明截短sgRNA降低了脱靶效应。
实施例3GSKO克隆传代稳定性验证结果
对鉴定出来的部分截短组GSKO克隆进行传代稳定性验证,使用CD CHO+6mMGlutaMAX以0.3的密度对GSKO克隆进行传代,每隔一天传一次代,结果如图2所示,可以看出GSKO克隆在15代内均正常生长增值,密度每天翻倍生长,活率一直维持在90%以上。
实施例4GSKO克隆表达蛋白应用
分别以29株GSKO克隆和CHO-K1对照为宿主细胞,采用电穿孔的方法稳定转染EGFP质粒,该质粒上连锁有外源性的GS基因。转染后在37℃、5%CO2培养箱中静置48h,换液,GSKO克隆仍使用不加MSX的CD CHO培养基,而CHO-K1细胞换成CD CHO+40μM MSX(Sigma-Aldrich,M5379)培养基。
筛选阶段如图3所示,流式分析仪(Beckman)检测EGFP阳性率,在换液后第2天,所有GSKO克隆细胞的EGFP阳性率均达到了90%以上。而CHO-K1细胞在换液后第5天EGFP阳性率才达到90%以上。除此之外,常规组GSKO-C23-EGFP和GSKO-C30-EGFP细胞在换液后第一天,EGFP阳性率较其他细胞低。总而言之,与CHO-K1宿主细胞相比,GSKO克隆极大地缩短了筛选所用的时间,且不需要使用MSX药物。而与常规组GSKO克隆相比,截短组GSKO克隆的筛选效果更好。
分别随机选择9株GSKO常规克隆和9株GSKO截短克隆为宿主细胞,采用电穿孔的方法稳定转染B0801质粒,筛选结束后,进行Fed-batch培养,通过D12收集培养上清,ELISA分析蛋白表达量,其常规组表达阶段的蛋白定量结果如图4所示,截短组表达阶段的蛋白定量结果如图5所示,截短组克隆蛋白表达量水平普遍高于常规组。此外,筛选出了4株显著高产GSKO克隆(GSKO-C22,GSKO-C2,GSKO-C4,GSKO-C6),其蛋白表达量高于未改造的CHO-K1细胞。其中截短组的GSKO-C2蛋白表达量最高,命名为中国仓鼠卵巢细胞CHOK1/GSKO-C2,2023年2月22日保藏于中国典型培养物保藏中心,保藏地址为中国武汉,武汉大学,保藏编号为CCTCC NO:C202342。
重组蛋白表达中代谢废物乳酸生成情况
除此之外,在Fed-batch过程中,每天收集培养上清,使用生化分析仪(西尔曼M9000)检测培养上清中产生的代谢废物乳酸含量,常规组表达阶段乳酸水平变化如图6所示,截短组表达阶段乳酸水平变化如图7所示。与CHO-K1对照相比,截短组GSKO克隆乳酸产生水平普遍低于对照,且后期乳酸消耗较快。与CHO-K1对照相比,常规组GSKO克隆乳酸产生水平或与对照持平,或低于对照。总的来说,GSKO克隆代谢废物乳酸生成量显著低于CHO-K1细胞。
Claims (9)
1.GS基因敲除的CHO细胞株构建方法,其特征在于,包括以下步骤:
(1)构建cas9circRNA和sgRNA:合成含有编码cas9蛋白的序列和附属元件的cas9线性化双链DNA,通过T7RNA聚合酶体外转录成线性化RNA,再将线性化RNA环化成circRNA;针对CHO细胞GS基因的CDS序列,设计sgRNA靶向序列,体外转录成靶向sgRNA;
(2)转染及流式分选:将cas9circRNA与sgRNA共转染CHO细胞,电转后流式分选;将存活的细胞进行分离,获得单克隆细胞,静置培养,得到单克隆细胞株;
(3)筛选与鉴定:将同一株单克隆细胞株中的一部分置于含有谷氨酰胺的培养基中培养,将一部分置于不含有谷氨酰胺的培养基中培养;在含有谷氨酰胺的培养基中能存活,但在不含谷氨酰胺的培养基中不能存活的细胞株,即为GS基因敲除的CHO细胞株,并通过westernblot进一步鉴定,无GS蛋白的细胞确定为GS基因敲除的CHO细胞株。
2.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(1)中,所述sgRNA靶向序列如SEQ ID NO.2所示。
3.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(1)中,sgRNA的构建是将sgRNA靶向序列与T7Promoter、gRNAscaffold(76bptracrRNA)和cPPT/CTS拼接成线性dsDNA,再体外转录成靶向sgRNA。
4.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(1)中,所述CHO细胞GS基因的CDS编码序列如SEQ ID NO.1所示。
5.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(2)中,电转总用量为10μg-30μg,优选20μg-30μg,最优选为20μg。
6.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(2)中,电转中cas9circRNA:sgRNA为1:2-5;优选1:2-4;最优选为1:3。
7.根据权利要求1所述的GS基因敲除的CHO细胞株构建方法,其特征在于,步骤(2)中,电转仪是Bio-rad,电转参数为250V,20ms,2次,5s,4mm。
8.一种GS基因敲除的中国仓鼠卵巢细胞CHOK1/GSKO-C2,2023年2月22日保藏于中国典型培养物保藏中心,保藏地址为中国武汉,武汉大学,保藏编号为CCTCCNO:C202342。
9.权利要求8所述GS基因敲除的CHO细胞株在重组蛋白表达中的应用。
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