Objective:To reconstruct pEGFP-C2-L539fs/47

a HERG nonsense mutant in eukaryotic expression plasmid

and observe the fusion protein expressed in HEK293 cells（human embryo kidney cells）.Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I

the small product fragment

from pcDNA3-L539fs/47

was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect

respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence

laser confocal imaging scanning in vivo

Western blot and PCR.Results:Mutation region cDNA fragment（about 1 kb） and target vector fragment（about 7.2 kb） were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47

constructed approximately 8.2 kb

sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD

the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells

which laid a foundation for the further study on L539fs/47.