中国生物工程杂志

The aim of the study was to construct the recombinant vector with double report gene of EGFP-LacZ which was expressed in eukaryotic cells. LacZ gene was amplified by PCR with pLenti6/V5-LacZ plasmid as template. The PCR products of LacZ gene were digested by restriction enzymes of EcoRⅠ and BamHⅠ, and linkaged with eukaryotic expression vector pEGFP-C1. The recombinant plasmid, named pEGFP-C1-LacZ, was identified by PCR and restriction analysis. pEGFP-C1-LacZ was thansfected into the eukaryotic cells of 293 FT and chicken embryo fibroblast (CEF) by lipofectin. The expression of EGFP-LacZ double report gene was detected by observing the green fluorescence and staining for beta -galactosidase activity. Green fluorescence was detected by fluorescence microscope in transfected cells. Moreover, the positive cell was observed by histochemistry of beta-galactosidase activity. The data indicated that pEGFP-C1-LacZ containing EGFP-LacZ double report gene has been constructed and expressed in eukaryotic cells successfully. The results would contribute to overcome the limitations and uncertainty caused by using of single report gene as molecular marker.