CHO细胞基因组NW-003614092.1内稳定表达位点的发现<sup>*</sup>

doi:10.13523/j.cb.2202035

中国生物工程杂志

2022, Vol. 42

Issue (6): 12-19 DOI: 10.13523/j.cb.2202035

研究报告

CHO细胞基因组NW-003614092.1内稳定表达位点的发现^*

瞿丽丽¹,丁学峰²,蔡燕飞¹,鲁晨¹,李华钟²,金坚¹,陈蕴^1,^**(

)

1.江南大学生命科学与健康工程学院无锡 214122
2.江南大学生物工程学院无锡 214122

Discovery of Stable Expression Sites in CHO Genome NW-003614092.1

QU Li-li¹,DING Xue-feng²,CAI Yan-fei¹,LU Chen¹,LI Hua-zhong²,JIN Jian¹,CHEN Yun^1,^**(

)

1. School of Life Science and Health Engineering, Jiangnan University, Wuxi 214122, China
2. School of Biotechnology, Jiangnan University, Wuxi 214122, China

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摘要：

目的:获得中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)内稳定表达位点信息,为构建重组蛋白CHO稳定表达株、缩短研发时间线提供信息明确的稳定位点。方法:对慢病毒随机整合Zsgreen1基因的具有潜在稳定表达位点的CHO-K1-1d2细胞株进行连续传代培养,验证表达稳定性;通过染色体步移分析慢病毒载体整合位点,并利用CRISPR/Cas9技术验证位点的可编辑性。结果:CHO-K1-1d2细胞在连续贴壁培养20代、悬浮培养50代过程中,能够100 %发绿色荧光,且荧光强度稳定,能够稳定表达Zsgreen1蛋白;染色体步移分析测序结果表明,CHO-K1-1d2细胞中慢病毒载体整合于CHO细胞基因组NW-003614092.1上第1 159 463与1 159 467碱基间;共转染sgRNA与Cas9质粒后,测序结果表明,该位点可被CRISPR/Cas9技术编辑。结论:CHO细胞基因组NW-003614092.1内存在一个信息明确的、能够被CRISPR/Cas9技术编辑的稳定表达位点。

关键词： CHO; 稳定位点; 染色体步移; CRISPR/Cas9

Abstract:

Objective: The purpose is to provide a stable expression site with clear information for constructing a Chinese hamster ovary cell (CHO) line stably expressing recombinant protein by site-specific integration and shortening the research and development timeline. Methods: The CHO-K1-1d2 cell line with potential stable expression site randomly integrated with Zsgreen1 gene by lentivirus was continuously subcultured to verify the stability of expression; the integration site of lentiviral vector was analyzed by chromosome walking, and the editability of the site was verified by CRISPR/Cas9 technology. Results: 100% of CHO-K1-1d2 cells could emit green fluorescence and the fluorescence intensity was stable in the process of continuous adherent culture for 20 generations and suspension culture for 50 generations, indicating that Zsgreen1 protein could be stably expressed. The sequence results in chromosome walking analysis showed that the antiviral vectors were integrated between bases 1 159 463 and 1 159 467 of the CHO cell genome NW-003614092.1. The sequence results after co-transfection of sgRNA and Cas9 plasmid in CHO-K1 cells showed that this site can be edited by CRISPR/Cas9 technology. Conclusion: There is a stable expression site in CHO cell genome NW-003614092.1, which has clear information and can be edited by CRISPR/Cas9 technology.

Key words: CHO Stable sites Genome walking CRISPR/Cas9

收稿日期: 2022-02-21 出版日期: 2022-07-07

ZTFLH:

Q813

基金资助: *国家高技术研究发展计划(2015AA020802)

通讯作者: 陈蕴 E-mail: chenyun72@126.com

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引用本文:

瞿丽丽,丁学峰,蔡燕飞,鲁晨,李华钟,金坚,陈蕴. CHO细胞基因组NW-003614092.1内稳定表达位点的发现^*[J]. 中国生物工程杂志, 2022, 42(6): 12-19.

QU Li-li,DING Xue-feng,CAI Yan-fei,LU Chen,LI Hua-zhong,JIN Jian,CHEN Yun. Discovery of Stable Expression Sites in CHO Genome NW-003614092.1. China Biotechnology, 2022, 42(6): 12-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2202035 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/12

表1 染色体步移引物序列

表2 位点验证引物序列

图1 整合位点验证示意图

图2 CHO-K1-1d2细胞贴壁培养20代Zsgreen1蛋白表达情况

图3 CHO-K1-1d2细胞悬浮培养50代Zsgreen1蛋白表达情况

图4 二级PCR产物琼脂糖凝胶电泳结果

表3 二级PCR产物测序结果

图5 整合位点验证PCR产物琼脂糖凝胶电泳结果

表4 位点验证测序结果

图6 位点可编辑性验证

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