CONSTRUCTION AND EXPRESSION OF EUKARYOTIC EXPRESSION VECTOR pcDNA3.1(+)-EGFP

Objective To construct the eukaryotic expression vector pcDNA3.1(+) EGFP and to detect transient expression of enhanced green fluorescent protein(EGFP) in laryngocarcinoma cells Hep 2. Methods According to the open reading frame of known EGFP gene, a pair of primers were designed and synthesized. A specific fragment of EGFP gene was obtained by PCR amplification from pAdTrack CMV containing EGFP gene.The PCR products were subcloned into pGEM T easy vector. Following digestion by HindⅢ/EcoRⅤ, the fragments of recombinants were cloned into the expression vector pcDNA3.1(+), then transformed into Escherichia coli DH5α, a recombinant pcDNA3.1(+) EGFP was constructed and confirmed by PCR and HindⅢ/EcoRⅤdigestion. It's transient expression was observed in 48 hours after it was transfected into laryngocarcinoma cells Hep 2. Results The eukaryotic expression vector pcDNA3.1(+) GFP was successfully constructed and GFP showing green fluorescence could be observed in laryngocarcinoma cells Hep 2 under fluorescent microscopy. Conclusion GFP was successfully expressed in laryngocarcinoma cells Hep 2. It was a good reporter and selection marker molecule in mammalian cells.