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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。
Objective To clone the complete P2X7 receptor coding region from leukemia cell line J6-1 and study its function. Methods The full-length P2X7 receptor coding sequence was amplified from J6-1 cells by RT-PCR and cloned into pTARGET eukaryotic expression vector and analyzed by DNA sequencing. The recombinant plasmid was transfected into HEK293 cells to obtain stable expression cell lines, RT- The expression of P2X7 receptor in HEK293 was detected by PCR and Western-blot. The formation of vesicles in transfected cells under agonist stimulation was observed by phase contrast microscopy. Results The complete sequence of the P2X7 receptor coding region was cloned from J6-1 cells and a sense mutation of A → G at position 559 resulted in Asn187 → Asp182. The P2X7 receptor derived from J6-1 cells can be expressed in HEK293 cells Expression, under the action of agonists can mediate the formation of vesicles. Conclusion The complete sequence of the P2X7 receptor coding region is cloned from leukemia cells. There may be other unknown mechanisms in J6-1 cells that result in the loss of P2X7 receptor function.