›› 2014, Vol. 41 ›› Issue (11): 93-96.

• 生物技术 • 上一篇    下一篇

猪NF-κB报告载体的构建及效率验证

李和刚, 张宝珣, 李培培, 杨培培, 江科, 孙友德, 戴正浩, 赵金山   

  1. 青岛市畜牧兽医研究所, 山东青岛 266100
  • 收稿日期:2014-05-26 出版日期:2014-11-20 发布日期:2014-12-06
  • 通讯作者: 赵金山 E-mail:zhaojinshande@sohu.com
  • 作者简介:李和刚(1980-),男,湖南人,博士,兽医师,研究方向:动物分子育种。
  • 基金资助:
    国家自然科学基金(31301936);青岛市科技计划基础研究项目(12-1-4-14-jch);山东省生猪产业技术体系;青岛市民生科技计划项目(14-2-3-45-nsh)。

Construction and Efficiency Detection of Porcine NF-κB Reporter Vector

LI He-gang, ZHANG Bao-xun, LI Pei-pei, YANG Pei-pei, JIANG Ke, SUN You-de, DAI Zheng-hao, ZHAO Jin-shan   

  1. Qingdao Institute of Animal Science and Veterinary Medicine, Qingdao 266100, China
  • Received:2014-05-26 Online:2014-11-20 Published:2014-12-06

摘要: 为开发出一套适用于猪NF-κB的报告系统,本试验利用猪IκBα基因启动子上6个NF-κB识别位点,设计构建了6个NF-κB报告载体(A~F)。通过转染猪髋动脉内皮细胞(PIEC)、猪肺泡巨噬细胞3D4/21及猪胎儿成纤维细胞,进行双荧光素酶检测。结果显示,本试验构建的6个报告载体(A~F)均能有效报告猪NF-κB p65亚基的过表达,且其荧光素酶活性上调倍数均高于pNF-κB-Luc载体;报告载体A~F的荧光素酶活性在3个细胞系中呈现出较为规律的差异,其中载体C活性最强,载体F活性最弱。结果表明,相对于商业化的NF-κB报告载体,所获得的6个载体(A~F)在上述3个细胞系中均表现出更为特异的转录活性,可作为猪物种特异的NF-κB报告载体使用。

关键词: NF-κB报告载体; 免疫; 猪; 特异性

Abstract: In order to provide a more dependent and fine tool for porcine immunology research, we developed a serial of pig specific NF-κB reporter system. Firstly, 6 reporter vectors were constructed by using 6 NF-κB recognition sites. Through transfection of porcine iliac endothelial cell line (PIEC), porcine alveolar macrophages cell line 3D4/21 and porcine primary fetal fibroblasts(CPEF), the relative luciferase expression was detected. These 6 reporter vectors, constructed in this study, could effectively report the expression of p65 subunit of porcine NF-κB, and the upregulation fold-changes were all higher than that of pNF-κB-Luc. The luciferase activities of reporter vectors A to F displayed regular difference among each other, of which reporter vector C showed the highest activities, while reporter vector F was the lowest. Compared to the commercial available NF-κB reporter vetor, those all 6 reporter vectors showed higher specificity of transcription activities. So our vector series could be used as NF-κB reporter vectors for the porcine species.

Key words: NF-κB reporter vector; immunity; porcine; specificity

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