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

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. CONCLUSIONS: 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.