Construction of the Prokaryotic Expression Vector of Green Fluorescent Protein

The green fluorescent protein(GFP)exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range and has no toxicity on host cells.In cell and molecular biology,the GFP is extensively used as a reporter or detection.In this paper,the GFP encoding sequence was proliferated by PCR under the direction of the primers designed according to the sequence obtained from NCBI website.Then the PCR product was inserted downstream the malE(maltose-binding protein)encoding gene between the EcoRⅠ and Hind Ⅲ sites after restriction endonuclease digestion,by which the gfp was fused with malE into one open reading frame.The recombinant prokaryotic expression vector of GFP was employed to transform the competent cells of E.coli TB1.The transformation mixture was cultured at 37 ℃ for 12-16 h on the LB plates with 50 mg/mL AMP and 10 μmol/L IPTG,and then cultured at 25-30 ℃ for 4-6 h on the same plate.The expression of GFP in TB1 was analysed by UV detection prior to SDS-PAGE.The GFP encoding gene infused dowmstream the malE gene can be expressed with green fluorescent excited by 365 nm UV under which the recombinant TB1 clone can be screened conveniently.Further analysis of SDS-PAGE showed that the GFP encoding gene was over expressed in E.coli host TB1.This result showed that the prokaryotic expression vector of GFP was successfully constructed and the malE fusion GFP could be expressed in high-level,which strongly supported the construction of the prokaryotic fusion expression vector with GFP as reporter and detection marker.