NF-κB荧光素酶报告基因系统的构建及验证

郭志兰1,2,3 车路阳4 李晶哲5 孙震晓1 刘长振5

(1.北京中医药大学中药学院, 北京 100102)
(2.中国中医科学院医学实验中心北京市重点实验室, 北京 100102)
(3.Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences, Beijing ,China 100700)
(4.中国人民解放军总医院骨科, 北京 100853)
(5.中国中医科学院医学实验中心北京市重点实验室, 北京 100700)

【摘要】为了定量检测NF-κB的活化效果及筛选与NF-κB活化调控相关的药物,通过去除逆转录病毒载体pQCXIP原有的CMV启动子,并分别插入NF-κB增强子序列及荧光素酶NanoLuc报告基因序列,构建了一种新的含有NF-κB增强子序列和NanoLuc(NLuc)报告基因序列的表达载体,并进一步建立受NF-κB调控的稳定表达NLuc荧光素酶的细胞系。酶切鉴定及测序结果表明成功构建了重组质粒pQCXIP-NF-κB-NLuc;NF-κB信号通路的刺激物肿瘤坏死因子TNF-α作用于构建的稳定表达NLuc的细胞系后出现特异性的荧光素酶反应,且该酶反应与TNF-α的刺激呈良好的时间、剂量依赖性,该结果表明受NF-κB调控的稳定表达NLuc荧光素酶的细胞系构建成功。实例验证中,NF-κB抑制剂雷公藤甲素对此细胞系NLuc荧光素酶表达的抑制呈剂量效应。综上,本实验构建的受NF-κB调控的稳定表达NLuc荧光素酶的报告基因系统可用于NF-κB的活化效果的定量检测及筛选与NF-κB活化调控相关的药物,具有研究和应用价值。

【关键词】 NF-κB; 荧光素酶NanoLuc; 报告基因系统;

【DOI】

【基金资助】 国家自然科学基金(Nos.31170829,81171762,81550017,81473418) National Natural Science Foundation of China(Nos.31170829,81171762,81550017,81473418) 中国中医科学院自主选题项目(No.zz2015015)资助 Research Project of CACMS(No.zz2015015)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1465-1473

October 2016

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摘要

  • 1 材料与方法
  • 2 结果与分析
  • 3 讨论
  • 参考文献