J6-1白血病细胞P2X7受体基因的克隆和功能初步分析

目的从白血病细胞系J6—1细胞中克隆P2X7受体编码区全序列并研究其功能。方法用RT—PCR法从J6—1细胞中扩增P2X7受体编码区全序列，克隆到pTARGET真核表达载体，并进行DNA序列分析；然后转染HEK293细胞，获得稳定表达细胞株，RT-PCR和Western blot法检测P2X7受体在HEK293中的表达；相差显微镜下观察转染细胞在激动剂刺激下膜泡的形成。结果从J6—1细胞中克隆了P2X7受体编码区全序列，在第559位有1个A—G有义突变，导致Asn187→Asp182；J6—1细胞来源的P2X7受体可在HEK293细胞中表达，在激动剂作用下可介导膜泡的形成。结论从白血病细胞中克隆出具有正常功能的P2X7受体编码区全序列，J6—1细胞中可能存在其它未知的机制造成P2X7受体功能丧失。

Cloning and functional analysis of P2X7 receptor from J6-1 leukemia cells

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.