先天性QT间期延长综合征相关基因SCN5A定点突变及蛋白表达研究  

Site-directed mutagenesis and protein expression of SCN5A gene associated with congenital Long QT syndrome

作　　者：史瑞明[1] 强华[1] 张艳敏[1] 马爱群[1] 高洁[1] 

机构地区：[1]西安交通大学医学院第一附属医院儿科,陕西西安710061

出　　处：《中国当代儿科杂志》2013年第3期223-226,共4页Chinese Journal of Contemporary Pediatrics

基　　金：国家自然科学基金(30900580)

摘　　要：目的构建先天性QT间期延长综合征相关基因SCN5A-delQKP1507-1509突变型真核表达载体,并观察其在人胚肾293(HEK293)细胞中的表达。方法采用一步法构建SCN5A-delQKP1507-1509突变型真核表达载体PEGFP-delQKP-hH1,用脂质体转染法将野生型和突变型质粒分别转染HEK293细胞,激光共聚焦显微镜观察钠通道蛋白在HEK293细胞的表达与定位,Western blot检测其蛋白表达。结果经电泳及DNA测序显示突变型1507-1509位点成功缺失9个碱基,野生型与突变型均在HEK293细胞膜上表达,且表达量差异无统计学意义(P>0.05)。结论成功构建钠通道基因SCN5A-delQKP1507-1509突变型真核表达载体,并在HEK293细胞中表达,为进一步研究其功能奠定基础。Objective To construct the sodium channel gene SCNSA-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney （HEK） 293 cells. Methods Eukaryotic expression vector PEGFP-delQKP-hH1 for SCN5AdelQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules. Results Direct sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells. Conclusions Successful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A protein in HEK293 cells provides a basis for further study on the functional effects of congenital long QT syndrome as a cause of SCN5A mutation.

关 键 词：先天性QT间期延长综合征 SCN5A基因 定点突变 真核表达载体 人胚肾293细胞 

分 类 号：R541.1[医药卫生—心血管疾病]