Construction of the eukaryotic vector carrying human dominant negative epidermal growth factor receptor,its expression and sub-cellular localization in COS-7 cells
AIM:To construct the eukaryotic expression vector (pEGFPN1-DNEGFR)carrying human dominant negative epidermal growth factor receptor(DNEGFR),detect the expression and the sub-cellular localization of dominant negative epidermal growth factor receptor-enhanced green fluorescence protein(DNEGFREGFP)in COS-7 cells transfected with pEGFPN1-DNEGFR.METHODS:The cDNA coding signal peptide,extracellular ligand-binding domain and membrane-spanning region of epidermal growth factor receptor(EGFR)precursor was obtained by reverse transcription-polymerase chain reaction(RT-PCR),then directionally cloned into the empty vector(pEGFP-N1)to construct pEGFPN1-DNEGFR.After confirmed by PCR amplification assay,double enzyme digestion,DNA sequencing and bioinformatics analysis of nucleotide sequence,pEGFPN1-DNEGFR was transfected into COS-7 cells,mediated by Lipofectamine 2000.The expression of and the sub-cellular localization of DNEGFREGFP in COS-7 cells were detected by Western Blot and Laser Scanning Spectral Confocal Microscope respectively.RESULTS:pEGFPN1-DNEGFR was constructed successfully,which was confirmed by PCR amplification assay,double enzyme digestion,DNA sequencing and bioinformatics analysis of nucleotide sequence.The expression of DNEGFR-EGFP was verified by Western Blot,and its predominant localization on the cell membrane of COS-7 cells was identified by Laser Scanning Spectral Confocal Microscope.CONCLUSION:The suecessful constrnction of pEGFPN1-DNEGFR,the expression and the sub-cellular localization of DNEGFR-EGFP in COS-7 cells,laid a solid foundation for further research on EGFR-targeted dominant negative strategy in cancer gene therapy.
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