CN110669791B - 一种提高细胞抗体表达量的方法 - Google Patents
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Abstract
本发明公开了一种提高动物细胞抗体表达量的方法。该方法包括以下步骤:(1)设计gRNA,具体序列如SEQ ID NO.1和SEQ ID NO.2所示;(2)将退火后的gRNA与质粒连接,并对连接后的质粒进行克隆保存;(3)采用步骤(2)所得质粒进行病毒包装,然后再对细胞进行病毒转染,培养5~7天后,筛选得到TRIM21蛋白敲除的单克隆细胞株,最后得到TRIM21蛋白敲除的细胞系。本发明通过敲除细胞内的TRIM21蛋白,可将细胞抗体表达量提高达20%‑50%,并且,在提高抗体表达量的同时不会影响抗体的质量,还能降低抗体的生产成本,缓解抗体市场的供需平衡。
Description
技术领域
本发明属于生物医学技术领域,具体涉及一种提高细胞抗体表达量的方法。
背景技术
抗体是指机体在抗原性物质刺激下,由B细胞分化成浆细胞所产生的、可与相应抗原发生特异性结合的免疫球蛋白。抗体针对相应抗原具有高特异性和高亲和力的特性,使得其在生物医学领域、疾病诊断和疾病治疗中有着无可比拟的优势。传统生产抗体的方法即利用抗原免疫动物后获得抗体,这种抗体称为多克隆抗体,即第一代抗体。80年代中期,随着DNA重组技术的发展以及人们对Ig分子认识的深入,抗体由单克隆抗体即第二代抗体发展到了第三代抗体:基因工程抗体。
基因工程抗体可以在多种体系成功表达,包括哺乳细胞、大肠杆菌、酵母细胞、转基因动植物等表达体系。然而因为哺乳细胞具有低免疫原性和且其细胞器可以提供最优的折叠、分泌和翻译后修饰的特点,使其成为治疗性抗体的首先细胞。但由于其生产成本高,构建周期长,基因操作困难,尤其是库存体积小导致生产的抗体量有限。近年来随着新的分子生物学技术、细胞工程及载体工程技术的快速发展,加之发酵技术的改进,这些都使抗体的表达量得到了提高。
到目前为止,提高抗体表达量的方法多为促进细胞株的代谢工程改造,并加强对细胞培养过程的理解和优化,提高抗体表达速率,开发高产量、高质量的细胞培养工艺。但是,无论是通过细胞工程或优化细胞培养来提高抗体表达量,其效率和产量都是有限的,而且并不是所有的蛋白都能在宿主细胞中获得很好的表达,加之人们对真核细胞表达调控机制认识不够充分造成抗体难于大规模化生产;另外,细胞工程技术生产的抗体成本极其昂贵(US$200/100mg)。由于基因工程抗体不易大规模化生产和高昂的生产成本从而制约了其临床应用。因此,亟需一种既能提高抗体表达量又不影响抗体安全性、特异性和纯度且能降低抗体生产成本的方法。
而TRIM蛋白(Tripartite motif family proteins)家族参与一系列的细胞生物学过程,人源TRIM约70多种。其中人源TRIM21蛋白的分子量为52KU,共含有475个氨基酸,由多个结构域组成。其中包含一个RING结构域、一个B-box结构域、一个coil-coil结构域以及C末端的B30.2结构域等。RING-Finger结构域是TRIM21作为泛素连接酶参与细胞代谢的基础,而其B30.2结构域能与IgG Fc段CH2-CH3结构域特异性结合。Clift D等人运用TRIM21蛋白的以上特性发明了Trim-away的方法,该方法是利用抗靶蛋白的IgG抗体靶向目标蛋白,再利用TRIM21靶向该IgG,从而特异性的敲除目的蛋白。然而目前未见关于通过敲除TRIM21来提高细胞抗体表达量的相关内容。
发明内容
针对现有技术中的上述不足,本发明提供一种提高细胞抗体表达量的方法,通过敲除细胞内TRIM21蛋白,能显著提高细胞抗体的表达量。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
一种提高细胞抗体表达量的方法,包括以下步骤:
(1)设计gRNA,具体序列如下:
gRNA-F:CACCGCAATCGACAGCTAGCCAACA;
gRNA-R:AAACTGTTGGCTAGCTGTCGATTGC;
(2)将退火后的gRNA与质粒连接,并对连接后的质粒进行克隆保存;
(3)采用步骤(2)所得质粒进行病毒包装,然后再对细胞进行病毒转染,培养5~7天后,筛选得到TRIM21蛋白敲除的单克隆细胞株,最后得到TRIM21蛋白敲除的细胞系。
进一步地,步骤(2)中gRNA的退火过程为:
gRNA-F和gRNA-R引物各取1μL,加水溶解至浓度为100μM,再补充8μL ddH2O,于95℃保持10min,然后以5℃/min退火至25℃。
进一步地,步骤(2)中gRNA与质粒的连接体系为:1μL T4连接酶、1μL退火后的gRNA、1μL 10×Ligase Buffer、50ng LentiCRISPRv2-puro,最后用ddH2O补足至11μL。
进一步地,细胞为哺乳动物细胞。
本发明的有益效果为:
(1)本发明通过敲除细胞内的TRIM21蛋白,可将细胞抗体表达量提高达20%-50%,并且,在提高抗体表达量的同时不会影响抗体的质量,例如抗体特异性、抗体滴度、抗体纯度等;还能降低抗体的生产成本,缓解抗体市场的供需平衡。
(2)该方法在提高抗体表达时具有通用性。
附图说明
图1为293T细胞敲除TRIM21后TRIM21表达量的WB检测结果图;
图2为敲除TRIM21对瞬时转染细胞内抗体表达量影响的WB检测结果图;
图3为敲除TRIM21对瞬时转染细胞培养液上清液中抗体表达量影响的WB检测结果图;
图4为构建的抗体稳定表达细胞系的细胞内以及培养液上清液中抗体表达量WB检测结果图;
图5为敲除TRIM21对抗体稳定表达细胞系的细胞内以及培养液上清液中抗体表达量影响的WB检测结果图。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
实施例1构建敲除TRIM21蛋白的293T细胞
1、构建gRNA表达载体
(1)根据网站https://www.genscript.com/gRNA-database.htmL提供的信息,获得TRIM21 CRISPR敲除gRNA:
gRNA-F:CACCGCAATCGACAGCTAGCCAACA;(SEQ ID NO.1)
gRNA-R:AAACTGTTGGCTAGCTGTCGATTGC;(SEQ ID NO.2)
(2)gRNA合成后,用水溶解引物成100μM,上下游各加1μL,补充ddH2O8μL,95℃,10min,5℃/min退火至25℃;LentiCRISPRv2-puro骨架载体经BsmBⅠ酶切,回收;酶切后骨架的质粒与退火后的gRNA在T4连接酶作用下16℃连接过夜,连接体系如表1所示:
表1连接体系
连接完成后,转化涂板挑选单个质粒克隆,测序检测质粒克隆核心序列的准确性,并摇菌提取大量质粒冻存。
(3)病毒包装
于100mm培养皿中复苏293T细胞,并将细胞密度培养至60%;然后分别在15mL的无菌管中配置DNA mixture和PEI mixture,其中:
DNA mixture包括1mL DMEM、4μg lentiCRISPR-puro-gDNA plasmid、3μg psPAX2packaging plasmid、1μg Pmd2.G envelope plasmid;
PEI mixture包括1mL DMEM和32μL PEI(1mg/mL)
然后将PEI mixture加入DNA mixture,轻轻混匀,室温静置15min,配置成transfaction mixture。
(4)吸弃293T原有培养基,加入transfaction mixture,并再加8mL DMEM至总培养体积10mL;随后放回细胞至培养箱,6h后换液;并在48h、72h各收一次培养基上清,然后用0.45um孔径的过滤器过滤,收集得到病毒上清;再将其暂时冻存于-80℃冰箱。
(5)病毒感染
将293T细胞接种于6孔板,细胞密度培养至60%;在无菌试管中配置1mL/孔新鲜完全培养基+4μL polybrene(4mg/mL);吸弃6孔板原有培养基,每孔加入配置的含有polybrene的培养基;摇匀后将细胞放回培养箱,37℃孵育1h;blank为lmL完全培养基,剩余每孔加入1mL 4℃解冻的病毒;病毒感染24h后换液,普通完全培养基继续培养48h;48h后换含有5μg/mL嘌呤霉素的完全培养基继续培养;每两天换一次液,待嘌呤霉素筛选一周后,挑取单克隆到96孔板培养,长满后扩大至48孔板继续培养,汇合度80%-90%时,消化取一半的细胞做裂解液PCR鉴定,剩余细胞原孔培养。
(6)TRIM21基因敲除细胞系的鉴定
细胞裂解液的PCR产物连T载送检测序;疑似阳性克隆细胞株系以RIPA裂解,Western Blot进一步鉴定TRIM21表达量,显示蛋白水平上TRIM21敲除成功(见图1)。
如图1所示,293T为野生型细胞株,D1~D9均为挑选的单克隆细胞株,在图1中,293T和D1~D9均在内参(GADPH)上具有清晰的条带,而293T、D2、D3和D4在TRIM21位置处呈现模糊的条带,D1、D5、D6、D7和D8甚至在TRIM21位置处不具有条带,表明在蛋白水平上TRIM21敲除成功。
实施例2检测TRIM21对体外瞬时转染抗体的表达量的影响
将293T(TRIM21+/+)、D1-293T(TRIM21-/-)、D2-293T(TRIM21-/-)分别以2×10^6个细胞的密度接种于6孔板中,培养24h;
然后将四对轻重链各2.5μg抗体表达质粒U111P1A6VH3-A6-1、U111P1A6K-A6-1;S12P2D4VH-1、S12P2D4K-6-1;S3P1B9VH3-B9-1、S3P1B9K-B9-2;S3P1H6VH3-H6-5、S3P1H6L3-H6-1共转于6孔板内,加无血清培养基,继续培养;48h后,收集细胞以及培养液上清,Western Blot检测细胞内(见图2)以及培养液上清液中(见图3)的抗体表达水平。
图2和图3中WT(TR+/+)表示293T(TRIM21+/+)细胞,即TRIM21未敲除细胞;D-1(TR-/-)和D-2(TR-/-)分别表示D1-293T(TRIM21-/-)细胞和D2-293T(TRIM21-/-)细胞,即TRIM21敲除细胞;根据图2和图3检测结果可知,在敲除了TRIM21后,细胞内的抗体表达水平明显升高,培养液上清液中的抗体表达水平同样也有明显升高。
实施例3构建抗体稳定表达且TRIM21敲除的细胞系
根据载体序列设计PCR引物克隆抗体重链或轻链序列,具体序列如下:
Ig retro-F:ATTGAATTCCACCATGGGATGGTCATGTATC;(SEQ ID NO.3)
Ig-H retro-R:CCATGGCGGCCGCAAGCTTTCCTCGTACGAC;(SEQ ID NO.4)
Ig-K retro-R:CCATGGCGGCCGCAAGCTTCTAACACTCTCC;(SEQ ID NO.5)
Ig-L retro-R:CCATGGCGGCCGCAAGCTTCTATGAACATTC;(SEQ ID NO.6)
利用高保真酶克隆抗体序列;用EcoR I和Not I分别酶切抗体序列克隆产物以及载体PMI-GFP、PMI-RFP,切胶回收;重链抗体序列与PMI-GFP,轻链抗体序列与PMI-RFP,在T4连接酶作用下过夜连接;连接体系转化涂板,挑取单克隆测序,阳性克隆进一步PCR、酶切鉴定;
分别以6μg pVPack-GP、6μg pVPack-VSV-G、6μg Ig H-PMI-GFP/6μg Ig L-PMI-RFP体系包装病毒,病毒上清多次感染293T(TRIM21+/+,作为对照)、D1-293T(TRIM21-/-,敲除TRIM21),流式分选GFP、RFP双阳性细胞,获得稳定表达三种抗体(S1PF1、S12P2D4和S35D4),且敲除TRIM21的单克隆细胞以及未敲除TRIM21的对照单克隆细胞;将单克隆细胞株系以RIPA裂解,然后Western Blot检测细胞内以及培养液上清液中的抗体,示例如图4;如图4所示,对Ig-H和Ig-L进行检测,分别检测出了S1PF1、S12P2D4和S35D4三种抗体的稳定表达。
实施例4检测TRIM21对抗体稳定表达细胞系的抗体表达量的影响
将S12P2D4-293T(TRIM21+/+、IgG+)、D11-S12P2D4-293T(TRIM21-/-、IgG+)细胞株分别以2×10^6个细胞的量接种于6孔板,培养48h,分别收集细胞和上清液,然后WesternBlot检测细胞内以及培养液上清液中抗体表达量变化,其检测结果见图5。
图5中左侧为细胞内抗体表达量的检测结果,右侧为培养液上清液中抗体表达量的检测结果,同时,图5中S12P2D4-293T(TR21+/+)表示未敲除TRIM21的且稳定表达S12P2D4抗体的293T(TRIM21+/+、IgG+)细胞;S12P2D4-293T-11(TR21-/-)表示敲除TRIM21的D11-S12P2D4-293T(TRIM21-/-、IgG+)细胞。
根据图5的检测结果可知,敲除TRIM21后,在稳转细胞内和培养液上清液中抗体的表达量均有明显升高。
序列表
<110> 成都益安博生物技术有限公司
<120> 一种提高细胞抗体表达量的方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccgcaatc gacagctagc caaca 25
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<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaactgttgg ctagctgtcg attgc 25
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
attgaattcc accatgggat ggtcatgtat c 31
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccatggcggc cgcaagcttt cctcgtacga c 31
<210> 5
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccatggcggc cgcaagcttc taacactctc c 31
<210> 6
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
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ccatggcggc cgcaagcttc tatgaacatt c 31
Claims (3)
1.一种提高细胞抗体表达量的方法,其特征在于,包括以下步骤:
(1)设计gRNA,具体序列如下:
gRNA-F:CACCGCAATCGACAGCTAGCCAACA;
gRNA-R:AAACTGTTGGCTAGCTGTCGATTGC;
(2)将退火后的gRNA与质粒连接,并对连接后的质粒进行克隆保存;
(3)采用步骤(2)所得质粒进行病毒包装,然后再对细胞进行病毒转染,培养5~7天后,筛选得到TRIM21蛋白敲除的单克隆细胞株,最后得到TRIM21蛋白敲除且S12P2D4抗体表达量提升的哺乳动物细胞系。
2.根据权利要求1所述的提高细胞抗体表达量的方法,其特征在于,步骤(2)中所述gRNA的退火过程为:
gRNA-F和gRNA-R引物各取1µL,加水溶解至浓度为100µM,再补充8µL ddH2O,于95℃保持10min,然后以5℃/min退火至25℃。
3.根据权利要求1所述的提高细胞抗体表达量的方法,其特征在于,步骤(2)中所述gRNA与质粒的连接体系为:1µL T4连接酶、1µL退火后的gRNA、1µL 10×Ligase Buffer、50ng LentiCRISPRv2-puro,最后用ddH2O补足至11µL。
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