中国当代儿科杂志2011，Vol.13Issue(8)：611-616,6.
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
摘要
Abstract
Objective To study the protocol of construction of a KCNQ2-c. 812G ＞ T mutant and it's eukaryotic expression vector, the c. 812G ＞ T (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. 812G ＞ T 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. 812G ＞ T 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. 812G ＞ T 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Key words
KCNQ2 gene/ Site-directed mutagenesis/ Eukaryotic expression vector分类
医药卫生引用本文复制引用
周熙惠,惠智艳,史瑞明,宋红霞,张葳,刘俐..新生儿惊厥相关基因KCNQ2定点突变及其蛋白表达的研究[J].中国当代儿科杂志,2011,13(8):611-616,6.基金项目
陕西省科学技术攻关项目(2007K14-05). （2007K14-05）
