新生儿惊厥相关基因KCNQ2定点突变及其蛋白表达的研究  

Site-directed mutagenesis and protein expression of KCNQ2 gene associated with neonatal convulsions

作　　者：周熙惠[1] 惠智艳[1] 史瑞明[1] 宋红霞[1] 张葳[1] 刘俐[1] 

机构地区：[1]西安交通大学医学院第一附属医院新生儿科/环境与疾病相关基因教育部重点实验室离子通道研究病室,陕西西安710061

出　　处：《中国当代儿科杂志》2011年第8期611-616,共6页Chinese Journal of Contemporary Pediatrics

基　　金：陕西省科学技术攻关项目(2007K14-05)

摘　　要：目的在收集的1个良性家族性婴儿惊厥家系中发现KCNQ2基因c.812G>T(p.G271V)突变,探讨c.812G>T突变体及其真核表达载体的构建方法,并观察其在人胚肾(HEK)293细胞中的表达。方法利用重叠延伸PCR和限制性内切酶法构建KCNQ2基因c.812G>T突变体真核表达载体pcDNA3.0-c.812G>T-KCNQ2,用脂质体转染法将KCNQ2质粒(野生型pcDNA3.0-KCNQ2或突变型pcDNA3.0-c.812G>T-KCNQ2)与荧光真核表达载体pRK5-GFP共转染HEK293细胞,激光共聚焦显微镜下观察HEK293细胞,免疫荧光化学法检测蛋白质表达。结果构建的突变体经DNA直接测序示KCNQ2基因cDNA第812位碱基G变为T,突变体在HEK293细胞中成功表达,野生型和突变型基因的蛋白质都在细胞膜上表达,提示KCNQ2钾通道孔区的突变c.812G>T并不影响蛋白的正常表达。结论成功构建KCNQ2突变基因真核表达载体,并在HEK293细胞中表达KCNQ2蛋白,为突变型KCNQ2基因的功能研究及癫癎的分子发病机制研究奠定基础。Objective To study the protocol of construction of a KCNQ2-c.812GT mutant and it′s eukaryotic expression vector,the c.812GT（p.G271V） mutation which was detected in a Chinese pedigree of benign familial infantile convulsions,and to examine the expression of mutant protein in human embyonic kidney（HEK） 293 cells. Methods A KCNQ2 mutation c.812GT was engineered on KCNQ2 cDNAs cloned into pcDNA3.0 by sequence overlap extension PCR and restriction enzymes.HEK293 cells were co-transfected with pRK5-GFP and KCNQ2 plasmid（the wild type or mutant） using lipofectamine and then subjected to confocal microscopy.The transfected cells were immunostained to visualize the intracellular expression of the mutant molecules. Results Direct sequence analysis revealed a G to T transition at position 812.The c.812GT mutation was correctly combined to eukaryotic expressive vector pcDNA3.0 and expressed in HEK293 cells.Immunostaining of transfected cells showed the expression of both the wild type and mutant molecules on the plasma membrane,which suggested that the c.812GT mutation at the pore forming region of KCNQ2 channel did not impair normal protein expression in HEK293 cells. Conclusions Successful construction of mutant KCNQ2 eukaryotic expression vector and expression of KCNQ2 protein in HEK293 cells provide a basis for further study on the functional effects of convulsion-causing KCNQ2 mutations and for understanding the molecular pathogenesis of epilepsy.

关 键 词：KCNQ2基因 定点突变 真核表达载体 

分 类 号：R720.597[医药卫生—急诊医学]