Li Yu, Cui Chang-cong, Huang Chen, Lian Jiang-fang, Zhao Yong-hui, Xue Xiao-lin, Zhao Xiao-ge
Department of Cardiology, First Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P. R. China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2006 Dec;23(6):627-30.
To investigate the protocol of the construction of HERG gene mutations, an A561V mutation which was detected in a Chinese congenital long QT syndrome (LQTS) family had been constructed and expressed in vitro.
The A561V cloning vector PGEM-HERG-A561V was constructed by quick site-directed mutagenesis PCR. The A561V expressive vector pcDNA3-HERG-A561V was constructed by restriction enzymes. pRK5-GFP was cotransfected with pcDNA3-HERG-A561V or wild type pcDNA3-HERG into HEK293 cells by Superfect transfection reagent. The protein was measured by immunofluorescence.
Direct sequence analyses revealed a C to T transition at position 1682. The A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation was expressed in cytoplasm and cellular membrane while the wild type gene was expressed only on cellular membrane.
The protocol can be used successfully to construct and express HERG A561V mutation and it forms the basement of the further study on functions of mutation.
为研究HERG基因突变的构建方法，构建了在中国一个先天性长QT综合征（LQTS）家系中检测到的A561V突变并在体外进行表达。
通过快速定点诱变PCR构建A561V克隆载体PGEM-HERG-A561V。通过限制性内切酶构建A561V表达载体pcDNA3-HERG-A561V。用Superfect转染试剂将pRK5-GFP与pcDNA3-HERG-A561V或野生型pcDNA3-HERG共转染入HEK293细胞。通过免疫荧光检测蛋白。
直接测序分析显示第1682位发生C到T的转变。A561V突变正确地整合到真核表达载体pcDNA3中并在HEK293细胞中表达。突变蛋白在细胞质和细胞膜中表达，而野生型基因仅在细胞膜上表达。
该方法可成功用于构建和表达HERG A561V突变，为进一步研究该突变的功能奠定了基础。
