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目的在收集的一个先天性长 QT 综合征家系中发现 HERG 基因 A561V 突变,探讨HERG 基因 A561V 突变在真核细胞中的功能表达。方法采用克隆载体快速 PCR 法及限制性内切酶法将突变体克隆到真核表达载体 pcDNA3中,用 Superfect 转染试剂将野生型及突变型 HERG 质粒与荧光载体 pRK5-GFP 共转染至 HEK293细胞,用免疫荧光化学法及蛋白免疫印迹法检测蛋白质表达,用全细胞膜片钳法检测 HERG 通道的电流表达。结果构建的突变体经 DNA 直接测序示HERG 基因 cDNAl682位点碱基 C 变为 T,突变体蛋白质位于细胞膜上及细胞质中,野生型通道蛋白显示135000和155000的2条蛋白条带,而杂合型和突变型通道蛋白仅有135000的1条蛋白条带,野生型通道记录到尾电流而杂合型通道及突变型通道均未检测到电流表达。结论成功构建并表达了 HERG 基因 A561V 突变的功能,中国患者具有与欧美患者相同的 HERG 基因突变热点。
Objective To detect the HERG gene A561V mutation in a collection of congenital long QT syndrome pedigrees to investigate the functional expression of the HERG gene A561V mutation in eukaryotic cells. Methods The mutant was cloned into eukaryotic expression vector pcDNA3 by cloning vector rapid PCR and restriction endonuclease. The wild-type and mutant HERG plasmids were co-transfected with pRK5-GFP and HEK293 Immunocytochemistry and Western blotting were used to detect the protein expression. The whole cell patch clamp method was used to detect the current expression of HERG channel. Results The direct DNA sequencing revealed that the base C of HER2 gene was changed to T and the mutant protein was located on the cell membrane and cytoplasm. The wild-type channel protein showed two protein bands of 135000 and 155000, while the heterozygous Type and mutant channel protein only 135000 a protein band, the wild-type channel to the tail current while the hybrid channel and the mutant channel were not detected current expression. Conclusion The function of HERG gene A561V mutation was successfully constructed and expressed. The Chinese patients had the same HERG gene mutation hot spot as the European and American patients.