[Cloning and functional analysis of P2X7 receptor from J6-1 leukemia cells]
Kun Nie1, Guo-Guang Zheng, Yong-Min Lin
1State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS & PUMC, Tianjin 30020, China.
Objective:
To clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells.
Methods:
The entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope.
Results:
The entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation.
Conclusions:
The entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.
