Objective:To construct a human ether-a-go-go-related gene(HERG)nonsense mutant L539fs/47-*558W into the autonomously fluorescent,eukaryotic expression vector pEGFP-C2,and to verify expression of the reconstruct in human embryonic kidney-293(HEK293)cells.Methods:The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of SbfⅠ,Eco91Ⅰand rejoining of T4 ligase.After verification,the recombinant pEGFP-C2-L539fs/47-*558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo.pcDNA3-L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce.The mutant protein size was determined by Western blotting.Results:The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery.pEGFP-C2-L539fs/47*-558W,approximately 8.2 kb,was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing.Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane,whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm,the others were transported to the cell membrane in living HEK293 cells.The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W.Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands.The mutant and mutant-GFP fusion proteins were 70 and 100 kDa,respectively.Conclusion:pEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells,which laid a foundation for the further study on L539fs/47-*558W. Objective: To construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs / 47- * 558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney -293 (HEK293) cells. Methods: The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of SbfI, Eco91I and rejoining of T4 ligase. AfterSolver, the recombinant pEGFP- C2- L539fs / 47- * 558W and pEGFP- C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. PcDNA3-L539fs / 47- * 558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results: The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs / 47- * 558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery. pEGFP-C2-L539fs / 47 * -558W, approximately 8.2 kb, was demonstrated successfully constructed constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was primarily expressed in the membrane , yet truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs / 47-558W.Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands.The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively.Conclusion: pEGFP-C2-L539fs / 47- * 558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs / 47- * 558W.