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

Abstract

ObjectiveTo 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). MethodsAfter 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. ResultsMutation 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. ConclusionspEGFP-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.