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Construction of a cho cell line with site‑specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

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Systems Microbiology and Biomanufacturing 2023年第4期3卷 659-668页

作者： Xuefeng Ding Yun Chen Hao Wu Zhaoqi Yang Yanfei Cai Yunfeng Dai Qinghe Xu Jian Jin Huazhong Li School of Biotechnology Jiangnan UniversityWuxiPeople’s Republic of China Department of Drug Design and Molecular Pharmacology School of Life Sciences and Health EngineeringJiangnan UniversityWuxiPeople’s Republic of China

Chinese hamster ovary(cho)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural ***,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random *** present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 *** results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of cho-K1 *** obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were *** study might provide a feasible method to construct a cho cell line with long-term stable expression of exogenous proteins.

关键词： cho Site-specific integration Stable expression CRISPR–Cas9 HSA

Selenium Regulation of Selenium-dependent Glutathione Peroxidases in Animals and Transfected cho Cells

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Biomedical and Environmental Sciences 1997年第2期10卷 346-355页

作者： ROGER A. SUNDE BRITTA M. THOMPSON MELANIE D. PALM SHERRI L.WEISS KEVIN M. THOMPSON AND JACQUELINE K. EVENSON(Nutritional Sciences Program and Department of Biochemistry,University of Missouri, Columbia MO 65211 USA) Nutritional Sciences Program University of Missouri Columbia 65211 USA.

Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with cho cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals

关键词： GPX mRNA Selenium Regulation of Selenium-dependent Glutathione Peroxidases in Animals and Transfected cho Cells cho

Electron spin resonance study on UV-irradiated single crystal of （NH3iPr）6[Mo8O28（cho）2]·2H2O

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Acta Chimica Sinica(English Edition) 1988年第4期6卷 308-314页

作者： CHEN Jie YOU Xiao-Zeng~* Institute of Coordination Chemistry,Nanjing University,NanfingHAN Shi-Ying SUI Yun-Xia The Centre of Material Analysis,Nanjing University,Nanjing Institute of Coordination Chemistry Nanjing University Nanjing The Centre of Material Analysis Nanjing University Nanjing

EPR study on UV-irradiated single crystal of（NH3iPr）6[Mo8O?（cho）2]·2H2O was carrie（?）out at room *** sets of EPR lines were *** Ⅰand Ⅱ are attributedrespectively to paramagnetic Mo（V）centre（A）and（B）.Line Ⅲ may be assigned to an unstableparamagnetic *** the least-squares-fitting method to calculate the Hamiltonian parameters,we got the accurate principal values of g,hyperfine AMo and ligand hyperfine AH tensors,as well asthe direction cosines of their principal axes with respect to experimental coordinate system（xyz）.Theelectron spin density was calculated from A *** results showed that the unpaired electronoccupies 4d orbital of molybdenum which appreciably contributes to the molecular orbital and themechanism of direct spin coupling between the unpaired spin orbital of molybdenum and hydrogenorbital.

关键词： Electron spin resonance study on UV-irradiated single crystal of Mo8O NH3iPr cho UV

表达重组蛋白的cho-GS细胞的无血清培养

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高技术通讯 2005年第7期15卷 73-78页

作者：张芳张立易小萍孙祥明张元兴华东理工大学生物反应器工程国家重点实验室上海200237

采用DOEHLERT和HADAMARD统计方法建立了一个适于重组cho-GS细胞生长和外源蛋白表达的无谷氨酰胺无血清培养基SFMB.对多种硫酸多聚糖进行了考察,选取低分子量硫酸葡聚糖,添加浓度为25 mg/L,可以避免细胞结团.用SFMB在方瓶中培养重组cho-G... 详细信息

采用DOEHLERT和HADAMARD统计方法建立了一个适于重组cho-GS细胞生长和外源蛋白表达的无谷氨酰胺无血清培养基SFMB.对多种硫酸多聚糖进行了考察,选取低分子量硫酸葡聚糖,添加浓度为25 mg/L,可以避免细胞结团.用SFMB在方瓶中培养重组cho-GS细胞,细胞生长优于含10%小牛血清的无谷氨酰胺DMEM/F12培养,最大细胞密度提高了40.7%,外源蛋白的表达水平也得到提高.在100 ml转瓶中分批培养,最大细胞密度和蛋白表达分别达到1.5×106 cells/ml和0.48 mg/L.

关键词：重组蛋白 HADAMARD DMEM/F12 无血清培养基谷氨酰胺蛋白表达细胞生长细胞密度统计方法低分子量添加浓度外源蛋白分批培养 cho 多聚糖葡聚糖牛血清 GS 硫酸转瓶

cho细胞基因组NW-003614092.1内稳定表达位点的发现

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中国生物工程杂志 2022年第6期42卷 12-19页

作者：瞿丽丽丁学峰蔡燕飞鲁晨李华钟金坚陈蕴江南大学生命科学与健康工程学院无锡214122 江南大学生物工程学院无锡214122

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... 详细信息

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1159463与1159467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

cho细胞基因组中稳定hot spot位点研究进展

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中国生物工程杂志 2022年第3期42卷 72-81页

作者：林健芬罗顺江西中医药大学南昌330004

近些年来,治疗性重组蛋白类药物是生物制药领域研究的热点。工业化生产中常用于重组蛋白表达的细胞系是中国仓鼠卵巢(Chinese hamster ovary,cho)细胞。传统cho细胞系的表达大多数基于随机整合的方式,这可能会使目标基因整合到异染色质... 详细信息

近些年来,治疗性重组蛋白类药物是生物制药领域研究的热点。工业化生产中常用于重组蛋白表达的细胞系是中国仓鼠卵巢(Chinese hamster ovary,cho)细胞。传统cho细胞系的表达大多数基于随机整合的方式,这可能会使目标基因整合到异染色质区域或者不稳定的染色质区域,导致cho细胞表达不稳定,需要多轮筛选才能获得理想的表达细胞系。最新研究表明,外源基因在cho细胞预测/特定的基因组位点中进行特异性整合,可以使重组cho细胞的表达保持长期一致性和稳定性。cho细胞基因组中高效稳定的转录整合位点被称为热点(hot spot)。阐述cho细胞基因组稳定的hot spot位点近几年的研究进展,其中包括热门的hot spot位点,以及如何研究新的hot spot位点的方法。总结如何将外源基因高效定位于预测的cho细胞hot spot位点,实现高水平稳定的表达重组蛋白,为发现新的有效的hot spot位点,构建稳定表达cho细胞系提供参考。

关键词： cho Hot spot 位点特异性整合细胞系开发

GRP75 of cho Cells Responds to Ribosylation

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生物化学与生物物理进展 2014年第11期41卷 1191-1192页

作者： LU Yang HE Rong-Qiao Institute of Biophysics Chinese Academy of SciencesBeijing 100101 China

Dear Editor,Recently,type 2diabetic patients were clinically observed to suffer from dysfunction not only in glucose metabolism,but also ribose[1-2].Ribose is rapidly reacted with protein[3-4],producing cytotoxic ribosylated products[5],whose progress is much faster than glucose[6-7].A 75 ku glucose regulated protein(GRP75)is generally recognized as a member of the heat shock protein 70(HSP70)class of proteins,which is induced under conditions of low glucose and other nutritional and environmental stresses[8-9].Administration of ribose induces a decrease in blood

关键词： GRP75 cho 生物化学生物物理

重组酶介导的定点整合及在构建重组cho细胞中的应用

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中国生物工程杂志 2022年第12期42卷 52-60页

作者：孙瑾瑜刘光李晨王颖刘国庆中国药科大学生命科学与技术学院南京211198 上海复宏汉霖生物技术股份有限公司上海200233 安腾瑞霖(上海)生物科技有限公司上海201620

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... 详细信息

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

全人源抗IL-33scFv-IgG1Fc融合蛋白在cho k1细胞中稳定高表达

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细胞与分子免疫学杂志 2016年第5期32卷 600-603,609页

作者：叶迎春年四季王栩吴桐徐文峰袁青四川医科大学基础医学院四川泸州646000

目的利用两种不同真核表达载体构建全人源抗白细胞介素33单链抗体c片段(IL-33 sc Fv-Fc)重组载体,转染中国仓鼠卵巢(cho)k1细胞,筛选稳定高表达单细胞株以提高融合蛋白产量。方法双酶切前期构建的重组质粒pc DNA3.1/SPsc Fv-Fc,回收SP-s... 详细信息

目的利用两种不同真核表达载体构建全人源抗白细胞介素33单链抗体c片段(IL-33 sc Fv-Fc)重组载体,转染中国仓鼠卵巢(cho)k1细胞,筛选稳定高表达单细胞株以提高融合蛋白产量。方法双酶切前期构建的重组质粒pc DNA3.1/SPsc Fv-Fc,回收SP-sc Fv-Fc片段,插入载体PMH3EN中,构建PMH3EN/SP-sc Fv-Fc重组载体并与重组质粒pc DNA3.1/SP-sc Fv-Fc分别转染cho k1细胞;反转录PCR、Western blot法检测目的基因sc Fv-Fc的转录及表达;Dot blot法筛选稳定高表达细胞株;ELISA检测sc Fv-Fc的水平及生物学活性。结果重组质粒测序结果表明成功构建了真核表达载体PMH3EN/SP-sc Fv-Fc,插入目的基因SP-sc Fv-Fc大小为1560 bp左右。反转录PCR检测重组质粒表达情况显示两组重组质粒的目的基因在cho k1细胞中均成功转录。sc Fv-Fc不仅可分泌到细胞培养基中,还可与人IL-33以及抗人Ig G1 Fc特异性结合。重组载体PMH3EN/SP-sc Fv-Fc组融合蛋白表达水平相对较高。经过4轮筛选,选出了稳定高表达细胞株,初步测其sc Fv-Fc表达产量为10 mg/L;竞争ELISA检测结果显示sc Fv-Fc可显著抑制IL-33与其受体ST2的结合。结论在cho k1细胞成功高表达IL-33 sc Fv-Fc。

关键词：白细胞介素33 单链抗体c片段(sc Fv-Fc) cho k1细胞表达载体