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中国应用生理学杂志 ›› 2016, Vol. 32 ›› Issue (5): 471-475.doi: 10.13459/j.cnki.cjap.2016.05.022

• 技术方法 • 上一篇    下一篇

人P2X7真核表达载体的构建及稳定转染细胞株的建立

魏林郁, 卢娜, 孟莉, 李新娟, 李璐, 李超堃, 李东亮   

  1. 新乡医学院基础医学院生理学与神经生物学研究室, 河南新乡 453003
  • 收稿日期:2016-02-29 修回日期:2016-05-30 出版日期:2016-09-28 发布日期:2018-06-20
  • 通讯作者: 李东亮,Tel:0373-3029104;E-mail:xyldl8@126.com E-mail:xyldl8@126.com
  • 基金资助:
    国家自然科学基金(81271376);河南省教育厅科学技术重点研究项目(14A310019,14A310009,16A310011);河南省基础与前沿技术研究计划资助项目(112300410164)

Construction of eukaryotic expressing vector of human P2X7 and establishment of stable transfectant cell line

WEI Lin-yu, LU Na, MENG Li, LI Xin-juan, LI Lu, LI Chao-kun, LI Dong-liang   

  1. Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, China
  • Received:2016-02-29 Revised:2016-05-30 Online:2016-09-28 Published:2018-06-20
  • Supported by:
    国家自然科学基金(81271376);河南省教育厅科学技术重点研究项目(14A310019,14A310009,16A310011);河南省基础与前沿技术研究计划资助项目(112300410164)

摘要: 目的:构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法:以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果:重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论:重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。

关键词: P2X7基因, 真核表达载体, 转染, HEK293细胞

Abstract: Objective: To construct eukaryotic expression vector of human P2X7gene and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods: P2X7 gene was amplified by polymerase chain reaction from the human brain P2X7 cDNA and inserted into a vector pEGFP-N1 to construct a recombinant plasmidcalled pEGFP-N1/P2X7. The correct recombinant plasmid was transfected into HEK293 cells by X-fect transfection reagent. The cell line stably expressing EGFP tagged-P2X7 gene were established by screening with G418 and fluorescence microscope. The expression levels and localization of human P2X7 in HEK293 cells was identified by flow cytometry, Western blot and laser scanning confocal microscope. Results: The recombinant plasmid pEGFP-N1/P2X7 was constructed correctly and the stable HEK293 cell line expressing EGFP tagged-P2X7 fusion protein was established. Both Western blot and flow cytometry revealed the higher expression of humanP2X7 in the stably transfected HEK293 cells. Under the laser scanning confocal microscope the EGFP tagged-P2X7 fusion protein was located on the membrane of HEK293 cells.Conclusion: The eukaryotic expressing vector of pEGFP-N1/P2X7 is successfully constructed and the HEK293 cell line stably expressing P2X7-EGFP fusion protein is established which have provided solid experimental foundation for further studies on the structure and function of P2X7 ionic channel.

Key words: P2X7 gene, eukaryotic expressing vector, transfection, HEK293 cell

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