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弱化抗性标记筛选高表达CHO细胞株的方法建立

刘苏, 田浤, 王驰, 姚文兵

刘苏, 田浤, 王驰, 姚文兵. 弱化抗性标记筛选高表达CHO细胞株的方法建立[J]. 中国药科大学学报, 2015, 46(5): 617-622. DOI: 10.11665/j.issn.1000-5048.20150517
引用本文: 刘苏, 田浤, 王驰, 姚文兵. 弱化抗性标记筛选高表达CHO细胞株的方法建立[J]. 中国药科大学学报, 2015, 46(5): 617-622. DOI: 10.11665/j.issn.1000-5048.20150517
LIU Su, TIAN Hong, WANG Chi, YAO Wenbing. Weakening resistance marker for establishing a process of screening high-producing CHO cell lines[J]. Journal of China Pharmaceutical University, 2015, 46(5): 617-622. DOI: 10.11665/j.issn.1000-5048.20150517
Citation: LIU Su, TIAN Hong, WANG Chi, YAO Wenbing. Weakening resistance marker for establishing a process of screening high-producing CHO cell lines[J]. Journal of China Pharmaceutical University, 2015, 46(5): 617-622. DOI: 10.11665/j.issn.1000-5048.20150517

弱化抗性标记筛选高表达CHO细胞株的方法建立

基金项目: 国家自然科学基金资助项目(No.81430082)

Weakening resistance marker for establishing a process of screening high-producing CHO cell lines

  • 摘要: 为了优化中国仓鼠卵巢细胞(CHO)表达体系,建立高表达CHO细胞株筛选方法,将外源基因表达载体上抗性筛选基因表达的新霉素磷酸转移酶(NPT)的261位氨基酸天冬氨酸突变成甘氨酸。经G418筛选,转染含突变型NPT表达载体的细胞存活率显著低于转染含野生型NPT表达载体的细胞存活率。以绿色荧光蛋白-抗体IgG1 Fc结构域融合蛋白为报告基因,验证了突变后新霉素磷酸转移酶对抗生素G418抗性减弱。经G418持续加压培养3周后,转染含突变型NPT表达载体的细胞中EGFP的表达量显著高于转染含野生型NPT表达载体的细胞,表明其具有筛选出高表达单克隆细胞株的潜力。
    Abstract: To optimize Chinese hamster ovary(CHO)expression system and establish a process of screening CHO cell lines with high productivity, neomycin-phosphotransferase(NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G. After selection by culturing with G418, the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bearing wide type NPT. An enhanced green fluorescent protein(EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene, which verified that the resistance of mutant-NPT to G418 was weakened. By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks, mutant-selected pools expressed more exogenous protein than the WT-selected pools. Therefore, the ratio of high producers in a transfected cell population greatly increased.
  • [1] Mingeot-Leclercq MP,Glupczynski Y,Tulkens PM.Aminoglycosides:activity and resistance[J].Antimicrob Agents Chemother,1999,43(4):727-737.
    [2] Sautter K, Enenkel B. Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity[J].Biotechnol Bioeng,2005,89(5):530-538.
    [3] Ho SC,Bardor M,Feng H,et al.IRES-mediated tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines[J].J Biotechnol,2012,157(1):130-139.
    [4] Cormack BP,Valdivia RH,Falkow S.FACS-optimized mutants of the green fluorescent protein(GFP)[J].Gene,1996,173(1):33-38.
    [5] Zhang C,Xing X.Fluorescent proteins as a visible molecular signal for rapid quantification of bioprocesses:potential and challenges[J].Chin J Chem Eng,2010,18(5):863-869.
    [6] Telford WG,Hawley T,Subach F,et al.Flow cytometry of fluorescent proteins[J].Methods,2012,57(3):318-330.
    [7] Wang Z,Chen Y,Li S,et al.Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells[J].Protein Expr Purif,2014,94(2):1-6.
    [8] Johnson DB,Xu J,Shen Z,et al.RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites[J].Nat Chem Biol,2011,7(11):779-786.
    [9] De Jesus M,Wurm FM.Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors[J].Eur J Pharm Biopharm,2011,78(2):184-188.
    [10] Kim JY,Kim YG,Lee GM.CHO cells in biotechnology for production of recombinant proteins:current state and further potential[J].Appl Microbiol Biotechnol,2012,93(3):917-930.
    [11] Xu X,Nagarajan H,Lewis NE,et al.The genomic sequence of the Chinese hamster ovary(CHO)-K1 cell line[J].Nat Biotechnol,2011,29(8):735-741.
    [12] Cacciatore JJ,Chasin LA,Leonard EF.Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system[J].Biotechnol Adv,2010,28(6):673-681.
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出版历程
  • 刊出日期:  2015-10-24

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