Construction of recombinant plasmid pEGFP-C2-L539fs/47 and its expression in HEK293 cells

Abstract 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 than 60%. 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. Conclusions 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.