Construction and expression of the recombinant containing carboxyl terminal sequence of L-type voltage-gated calcium channel α_(1C) subunits

Objective To construct and express in COS-7 cells the enhanced green fluorescent protein(EGFP) tagged carboxyl terminal sequence(CT,1587-2169) of L-type voltage-gated calcium channel α1C subunits.Methods The CT cDNA was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1.EGFP cDNA was amplified by PCR and cloned into pcDNA3.1-CT recombinant.EGFP was tagged to the C-terminus of the full-length CT.The recombinant was transfected to COS-7 cells by the method of lipofection and the protein expression was evaluated by fluorescence microscopy and immunoblot analysis.Results CT cDNA and EGFP cDNA were cloned.The pcDNA3.1-CT-EGFP recombinant constructed was verified by restriction endonuclease digestion and sequencing.Green fluorescence was shown in COS-7 cells transfected with the recombinant.The immunoblot analysis displayed a distinct band in the pcDNA3.1-CT-EGFP transfected group.Conclusion pcDNA3.1-CT-EGFP recombinant is successfully constructed and can be expressed in COS-7 cells.