长QT综合征KCNQ1基因G983A突变体的构建、体外转录与表达  

Construction,in vitro transcription and expression of KCNQ1(G983A) gene mutant

作　　者：李伟[1] 杨钧国[2] 杜戎[2] 管思明[1] 柯琴梅[1] 王斌[1] 徐秋梅[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院老年病科,湖北武汉430022 [2]华中科技大学同济医学院附属协和医院心血管病研究所,湖北武汉430022

出　　处：《心脏杂志》2008年第6期673-677,共5页Chinese Heart Journal

基　　金：国家自然科学基金项目资助(30170377);国家科技攻关计划(863)项目资助(2002BA711A07)

摘　　要：目的构建先天性长QT综合征KCNQ1基因G983A突变体,并研究其在非洲爪蟾卵母细胞上的表达。方法首先进行定点诱变,构建含KCNQ1G983A突变点的原核表达载体pSP64-KCNQ1(G983A),并对pSP64-KCNQ1(G983A)进行纯化和线性化,然后应用SP6RNA体外转录试剂盒进行转录,并回收和鉴定转录的cRNAs。制备非洲爪蟾卵母细胞,经显微注射仪将KCNQ1(G983A)cRNAs注射至卵母细胞中,2d后采用双电极电压钳记录卵母细胞膜的电流。结果成功地构建pSP64-KCNQ1(G983A)突变体,经转录获得KCNQ1(G983A)cRNAs,并在爪蟾卵母细胞上表达。结论KCNQ1基因(G983A)突变体的成功构建及表达,为进一步探讨长QT综合征的发病机制奠定了基础。AIM To construct the vector of KCNQ1 G983A mutation and to investigate its expression in Xenopus laevis oocytes. METHODS Site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. pSP64-KCNQ1 （G983A） was purified and linearized by digestion, and eRNAs were then prepared with SP6 RNA polymerase, cRNAs were injected to Xenopus laevis oocytes by microinjectors. Two days later, the membrane currents of oocytes were recorded by dual-microelectrode voltage-clamp technique. RESULTS The mutation site in pSP64-KCNQ1 （G983A） was correctly established and pSP64-KCNQ1 （G983A） was transcribed in vitro and expressed in Xenopus laevis oocytes successfully. CONCLUSION The successful construction and expression of KCNQ1 G983A lay the foundation for further study on the mechanism of long QT syndrome.

关 键 词：长QT综合征 KCNQ1基因 突变体 构建 表达 

分 类 号：Q343.1[生物学—遗传学]