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目的:建立稳定的先天性长QT综合征相关HERG基因的细胞株,并观察其蛋白质表达。方法:原核克隆载体PGEM-HERG经限制性内切酶获得HERG cDNA,将HERG cDNA亚克隆到真核表达载体pcD-NA3中,用Lipofectamin2000转染试剂介导将pcDNA3-HERG及荧光真核表达载体PRK5-GFP共转染至HEK-293细胞,利用G-418进行细胞筛选,并用稀释法建立稳定的HEK-HERG细胞株,用细胞免疫荧光化学法及蛋白免疫印迹法(Western-blot)检测基因的蛋白质表达。结果:建立的HEK-HERG细胞株稳定传代,细胞免疫荧光化学法及Western-blot法检测到了HERG通道蛋白质的表达。结论:该方法可成功建立HEK-HERG细胞株并表达HERG通道蛋白质,为今后突变型HERG基因的研究提供了细胞基础。
OBJECTIVE: To establish a stable cell line of congenital long QT syndrome-associated HERG gene and observe its protein expression. METHODS: The prokaryotic cloning vector pGEM-HERG was obtained by restriction endonuclease to obtain HERG cDNA. The HERG cDNA was subcloned into eukaryotic expression vector pcD-NA3 and transfected with pcDNA3-HERG and the fluorescent eukaryotic expression vector PRK5-GFP was co-transfected into HEK-293 cells, G-418 cells were used for screening, and stable HEK-HERG cell lines were established by dilution. The gene was detected by immunofluorescence and Western-blot Protein expression. Results: HEK-HERG cell line established stable passage, HERG channel protein expression detected by immunofluorescence and Western-blot. Conclusion: This method can successfully establish HEK-HERG cell line and express HERG channel protein, which provides a cellular basis for the future study of mutant HERG gene.