先天性长QT综合征相关HERG基因细胞株的建立及其蛋白质表达

Construction of cell strain of congenital long QT syndrome related HERG gene and its protein expression

目的:建立稳定的先天性长QT综合征相关HERG基因的细胞株,并观察其蛋白质表达。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pcD-NA3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细胞株,用细胞免疫荧光化学法及蛋白免疫印迹法(Western-blot)检测基因的蛋白质表达。结果:建立的HEK-HERG细胞株稳定传代,细胞免疫荧光化学法及Western-blot法检测到了HERG通道蛋白质的表达。结论:该方法可成功建立HEK-HERG细胞株并表达HERG通道蛋白质,为今后突变型HERG基因的研究提供了细胞基础。

Objective：To construct the cell strain of Congenital Long QT syndrome related HERG gene and investigate its protein expression. Method： 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 strain was set up by dilution methods. The protein of HERG was measured by immunofluorescence and Western-blot. Result： HERG was correctly combined to eukaryotic expression vector pcDNA3. 293 cells. The stable HEK-HERG cell strain was maintained in selection medium and protein was expressed. Conclusion：The protocol can be used successfully to construct HEK-HERG and express H ERG protein. This form the basement of the further study on HERG mutations.