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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

彭冰, 岳军, 杜彩萍, 侯筱宇

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Abstract

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.

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What this paper is about

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.

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Available abstract

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.

Key concepts: Recombinant DNA, Complementary DNA, Molecular biology, Green fluorescent protein, Transfection, Restriction enzyme, Biology, Plasmid

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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 — Research Paper | ScholarLens