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.