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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (12): 52-60    DOI: 10.13523/j.cb.2207027
综述     
重组酶介导的定点整合及在构建重组CHO细胞中的应用
孙瑾瑜1,2,刘光2,李晨3,王颖1,*(),刘国庆2,*()
1 中国药科大学生命科学与技术学院 南京 211198
2 上海复宏汉霖生物技术股份有限公司 上海 200233
3 安腾瑞霖(上海)生物科技有限公司 上海 201620
Recombinase-mediated Site-specific Integration and Its Application in the Construction of Recombinant CHO Cells
SUN Jin-yu1,2,LIU Guang2,LI Chen3,WANG Ying1,*(),LIU Guo-qing2,*()
1 College of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China
2 Shanghai Henlius Biotech, Inc., Shanghai 200233, China
3 Aton (Shanghai) Biotech Co., Ltd., Shanghai 201620, China
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摘要：

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)是生产治疗性重组蛋白最常用的细胞。随机整合(random integration,RI)工艺是目前构建重组CHO细胞株的主要策略,由于CHO细胞基因组缺乏稳定性,为得到产量高、品质好且适应特定工艺的细胞株,通常需要1~2轮高通量筛选,不仅工作量大、耗时长且批次稳定性差。定点整合(site-specific integration,SSI)基因编辑技术将外源基因整合至细胞基因组的特定位点,经一轮筛选得到稳定、高产且适应特定生产工艺的细胞株,从而缩短细胞株构建周期。近年来,不断有将定点整合策略应用于构建重组CHO细胞株的报道。基因编辑技术的发展是实现细胞外源基因定点整合工艺的基础,常用的基因编辑技术包括核酸酶技术、转座子技术和重组酶技术。比较这三种基因编辑技术在构建流程、整合效率和专利等方面的不同特点,重点讨论重组酶介导的定点整合及其在CHO细胞株构建中的应用。
关键词： 基因编辑;  定点整合;  重组酶;  CHO;  重组酶介导盒式交换    
Abstract:

Chinese Hamster Ovary (CHO) cells are the most commonly used cells for producing therapeutic recombinant proteins. At present, random integration (RI) is still the main strategy for the construction of recombinant CHO cell lines. Due to the lack of genomic stability in CHO cells, 1-2 rounds of high-throughput screening are usually required to obtain cell lines with high yield, good quality and suitable for a specific process,which is a process with heavy workload, time-consuming and poor batch stability. Site-specific integration (SSI) is a gene editing technique that integrates foreign genes into specific sites to obtain stable, high-yield cell lines adapted to specific production process through one round of screening, thus shortening the construction cycle of cell lines. In recent years, there have been continuous reports of cases where the site-specific integration strategy was applied to construct recombinant CHO cell lines. The most commonly used techniques are nuclease, transposon, and recombinase techniques. The recombinase with great commercial prospect and its application in CHO cell line construction are discussed after considering the construction process, efficiency and patents.
Key words: Gene editing    Site-specific integration    Recombinase    CHO    Recombination-mediated cassette exchange (RMCE)
收稿日期: 2022-07-14 出版日期: 2023-01-05
ZTFLH:  Q819  
通讯作者: 王颖,刘国庆     E-mail: waying@CPU.edu.cn;Guoqing_Liu@henlius.com
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引用本文:

孙瑾瑜,刘光,李晨,王颖,刘国庆. 重组酶介导的定点整合及在构建重组CHO细胞中的应用[J]. 中国生物工程杂志, 2022, 42(12): 52-60.

SUN Jin-yu,LIU Guang,LI Chen,WANG Ying,LIU Guo-qing. Recombinase-mediated Site-specific Integration and Its Application in the Construction of Recombinant CHO Cells. China Biotechnology, 2022, 42(12): 52-60.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2207027    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I12/52

图1  重组酶介导的整合、切除和倒置反应
类型 脱靶率 位点依赖 构建过程 商业专利归属
核酸酶技术 ZFNs技术 高 不依赖 复杂 Sangamo
TALENs技术 高 不依赖 较复杂 Cellectis Bioresearch
CRISPR/Cas9技术 低 不依赖 简便 Broad Institute、Berkeley、Merck KGaA,等
转座子技术 - 低 依赖 简便 ATUM、Lonza、ProBioGen
重组酶技术 - 低 依赖 简便 Applied Stem Cell
表1  三种基因编辑技术比较
图2  酪氨酸重组酶和丝氨酸重组酶介导的RMCE
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