通用型NF-κB荧光素酶报告基因细胞系的构建

doi:10.13560/j.cnki.biotech.bull.1985.2019-0058

生物技术通报 ›› 2019, Vol. 35 ›› Issue (7): 129-133.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0058

通用型NF-κB荧光素酶报告基因细胞系的构建

郑海亮¹, 王东方¹, 方雅¹, 刁小伟¹, 文勇^1,2, 袁云霞^1,2

1. 江苏奥赛康生物医药有限公司,南京 211112;
2. 江苏奥赛康药业股份有限公司,南京 211112

收稿日期:2019-01-17 出版日期:2019-07-26 发布日期:2019-07-29
作者简介:郑海亮,男,硕士,研究方向：药理学、生物制品开发;E-mail：zhenhailiang@ask-pharm.com

Establishment of NF-κB Luciferase Reporter Cell Line with Commonality

ZHENG Hai-liang¹, WANG Dong-fang¹, FANG Ya¹, DIAO Xiao-wei¹, WEN Yong^1,2, YUAN Yun-xia^1,2

1. Jiangsu Aosaikang Biopharmaceutical Co.,Ltd.,Nanjing 211112;
2. Jiangsu Aosaikang Pharmaceutical Co.,Ltd.,Nanjing 211112

Received:2019-01-17 Published:2019-07-26 Online:2019-07-29

摘要/Abstract

摘要： 建立一种通用型荧光素酶报告基因检测系统。以NF-κB信号通路为基础,用NF-κB-RE-Luc2P慢病毒感染 HEK293细胞获得单克隆细胞系,并使用TNF-α进行了刺激验证;为证明该细胞系具备通用和改造能力进一步感染了FGF家族受体FGFRⅡ,筛选获得单克隆然后用FGF类药物进行刺激验证。结果表明,在TNF-α刺激实验中,响应值与TNF-α浓度呈现剂量依赖效应,且信噪比可达100倍以上,表明细胞系建立成功。FGF类药物刺激验证实验中,响应值与药物浓度可拟合成剂量依赖的“S”型曲线,表明该细胞系可用于FGF类药物活性检测。获得了NF-κB-RE-Luc2P HEK293和以此细胞系为基础改造得到的细胞系FGFRⅡ/NF-κB-RE-Luc2P HEK293,由于NF-κB是细胞内众多信号通路的交汇点,因此针对NF-κB信号通路,该细胞系具备通用性。

关键词: NF-KB, FGFRⅡ, 通用型, 荧光素酶报告基因

Abstract: This work is aimed to establish luciferase reporter gene system with commonality. Lentivirus containing NF-κB-RE-Luc2P was transduced into HEK293 and monoclonal HEK293 cell line expressing luciferase reporter gene under control of NF-κB response element was established and verified with TNF-α stimulation. To demonstrate the cell line capacity of commonality and modification,FGF family receptor FGFRⅡwas further transduced into this cell line and the screened FGFRⅡ/NF-κB-RE-Luc2P HEK293 single clones was further validated by FGF analogue stimulation. Dose-response effect and more than 100 fold of signal to noise ratio were observed in this cell line in TNF-α stimulation,indicating the cell line was generated successfully. In response to FGF analogue stimulation,dose-response effect was fitted as the “S” curve,implying that the cell line can be applied to the activity detection of FGF-type medicine. The results showed that NF-κB-RE-Luc2P HEK293 and modified one FGFRⅡ/NF-κB-RE-Luc2P HEK293 was established. that the cell line has commonality regarding NF-κB signaling.

Key words: NF-κB;, FGFRⅡ, commonality, luciferase reporter gene

郑海亮, 王东方, 方雅, 刁小伟, 文勇, 袁云霞. 通用型NF-κB荧光素酶报告基因细胞系的构建[J]. 生物技术通报, 2019, 35(7): 129-133.

ZHENG Hai-liang, WANG Dong-fang, FANG Ya, DIAO Xiao-wei, WEN Yong, YUAN Yun-xia. Establishment of NF-κB Luciferase Reporter Cell Line with Commonality[J]. Biotechnology Bulletin, 2019, 35(7): 129-133.

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通用型NF-κB荧光素酶报告基因细胞系的构建

Establishment of NF-κB Luciferase Reporter Cell Line with Commonality

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