L型电压门控钙通道α(下标 1C)亚基羧基末端肽段真核表达载体的构建和表达

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 α(subscript 1C) 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 hand 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.