人P2X7真核表达载体的构建及稳定转染细胞株的建立

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

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P2X7c DNA为模板扩增出P2X7基因,插入到真核表达载体p EGFP-N1中,构建重组质粒p EGFPN1/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p EGFP-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p EGFP-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。

Objective: To construct eukaryotic expression vector of human P2X7 gene 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 c DNA and inserted into a vector p EGFP-N1 to construct a recombinant plasmid called p EGFP-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 p EGFP-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 human P2X7 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 p EGFP-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.