BAK与BAX敲除型CHO细胞生物学性状的研究

曹啸东; 蔡燕飞; 金坚

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BAK与BAX敲除型CHO细胞生物学性状的研究

首发时间：2020-06-05

曹啸东 ¹
曹啸东（1993-），男，硕士研究生，主要研究方向：生物制药
蔡燕飞 ¹ 金坚 ¹
金坚（1960-），男，博士，教授，研究方向：肿瘤耐药机制研究

1、江南大学药学院药物设计与分子药理实验室，无锡 214122

摘要：敲除CHO细胞中BAK/BAX基因是抗CHO细胞凋亡的策略之一，该方法在提高目标蛋白表达产量、简化纯化步骤方面意义重大。然而目前其应用并不广泛，主要原因可能在于对BAK/BAX敲除型细胞的生物学性状研究不够深入。本文运用研究室前期构建的BAK单敲除与BAK/BAX双敲除CHO-K1细胞，首先通过批次和流加培养了解细胞的生长情况，然后运用不同类型的促凋亡药物作用于细胞，通过MTT药敏性实验和流式凋亡实验检测敲除型细胞与野生型细胞的抗凋亡情况，以期为BAK/BAX敲除型CHO细胞的应用提供思路。实验结果表明：（1）与野生型CHO-K1细胞比较，BAK单敲除细胞与BAK/BAX双敲除细胞能够耐受嘌呤霉素和丁酸钠诱导的细胞凋亡。表明BAK/BAX敲除型CHO-K1细胞能够在一定程度上抑制细胞通过内质网途径和细胞核途径发生细胞凋亡，并提示可通过添加丁酸钠提高目标蛋白的表达水平；（2）BAK单敲除与BAK/BAX双敲除两种细胞的抗凋亡能力相当，提示可能BAK单敲除即可达到期望的抗凋亡效果；（3）BAK/BAX敲除型细胞对G418较为敏感，提示G418可作为BAK/BAX敲除型外源基因表达细胞克隆筛选的抗性标记。本研究通过进一步检测BAK单敲除与BAK/BAX双敲除CHO-K1细胞的生物学性状，为其后期的研究与工业应用提供借鉴。

关键词：生物制药 BAK/BAX基因 CHO细胞抗凋亡

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Study on the biological characteristics of BAK and BAX knockout CHO cells

Cao Xiaodong ¹
曹啸东（1993-），男，硕士研究生，主要研究方向：生物制药
Cai Yanfei ¹ Jin Jian ¹
金坚（1960-），男，博士，教授，研究方向：肿瘤耐药机制研究

1、School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China

Abstract：Knocking out the BAK/BAX gene in CHO cells is one of the strategies to resist CHO cell apoptosis. This method is of great significance in improving the expression yield of target protein and simplifying the purification steps. However, its application is not widely at present, and the main reason may be that the biological characteristics of BAK/BAX knockout cells are not deeply studied. In this paper, the BAK single knockout and BAK/BAX double knockout CHO-K1 cells constructed in the early stage of the laboratory are used to research, firstly study the growth of cells through batch and feed culture, and then use different types of pro-apoptotic drugs to induce cells apoptosis. The anti-apoptotic situation of knock-out cells and wild-type cells was detected by MTT drug sensitivity experiment and flow cytometry experiment, in order to provide ideas for the application of BAK/BAX knock-out CHO cells. The experimental results show that: (1) Compared with wild-type CHO-K1 cells, BAK single knockout cells and BAK/BAX double knockout cells can tolerate apoptosis induced by puromycin and sodium butyrate. Shows that BAK and BAX knockout CHO-K1 cells can inhibit the apoptosis of cells through endoplasmic reticulum pathway and nuclear pathway to a certain extent, and suggests that the expression level of target protein can be increased by adding sodium butyrate; (2) BAK single knockout and BAK/BAX double knockout cells have the same anti-apoptotic ability, suggesting that BAK single-knockout can achieve the desired anti-apoptotic effect; (3) BAK/BAX knock-out cells are more sensitive to G418 , suggesting that G418 can be used as a resistance marker for the screening of clones of BAK/BAX knock-out foreign gene expression cells. In this study, by further testing the biological characteristics of BAK single knockout and BAK/BAX double knockout CHO-K1 cells, it can provide a reference for its later research and industrial application.

Keywords： Biological pharmaceutical BAK/BAX gene CHO cells Anti-apoptosis

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曹啸东，蔡燕飞，金坚. BAK与BAX敲除型CHO细胞生物学性状的研究[EB/OL]. 北京：中国科技论文在线 [2020-06-05]. https://www.paper.edu.cn/releasepaper/content/202006-30.

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论文编号	202006-30
论文题目	BAK与BAX敲除型CHO细胞生物学性状的研究
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