先天性长QT综合征相关人类HERG基因真核表达载体的构建及其功能表达

目的构建先天性长QT综合征相关HERG基因的真核表达载体,观察其在真核细胞中的表达,建立稳定的细胞系,探讨基因的功能。方法原核克隆载体pGEM-HERG经限制性内切酶获得HERG cDNA,将HERG cDNA亚克隆到真核表达载体pcDNA3中,用Lipofectamin2000转染试剂介导将pcDNA3-HERG及荧光真核表达载体pRK5-GFP共转染至HEK-293细胞,利用G-418进行细胞筛选,并用稀释法建立稳定的HEK-HERG细胞系。用全细胞膜片钳技术检测HERG基因的功能表达情况。结果在原核克隆载体pGEM-HERG的基础上构建了HERG的真核表达载体pcDNA3-HERG,并使其在HEK-293细胞中成功表达,建立的HEK-HERG细胞系稳定传代。膜片钳技术检测到了HERG通道电流的表达。结论该方法可成功构建及表达HERG的真核表达载体,为今后突变型HERG的研究奠定了基础。

Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene and investigation of its function expression

Objective To construct the eukaryotic vector of congenital long QT syndrome related HERG gene and investigate its expression in eukaryotic cell,and thus to set up a stable cell line of HERG.Methods HERG cDNA was obtained from pGEM-HERG by restriction enzymes.HERG cDNA was subcloned into pcDNA3 vector to gain HERG eukaryotic expression vetor pcDNA3-HERG.pcDNA3-HERG and pRK5-GFP were cotransfected into HEK 293 cells.Transfected cells were selected by geneticin(G-418).Stable HERG cell line was set up by dilution methods.The function expression of HERG was measured by whole-cell patch-clamp techniques.Results HERG was correctly combined to eukaryotic expression vector pcDNA3 and expressed in HEK 293 cells.The stable HERG-HEK cell line was maintained in selection medium.HERG current was detected by patch-clamp.Conclusion The protocol can be used to successfully construct eukaryotic expression vector of HERG and express HERG function.This is the foundation of the further study on HERG mutations.