EGFP-LacZ双报告基因真核表达载体的构建及体外表达

Construction of the Expression Vector with Double Report Gene of EGFP-LacZ and Its Expression in Vitro

构建(β-半乳糖苷酶与增强型绿色荧光蛋白(EGFP)双报告基因的真核表达载体,并在真核细胞中表达。采用PCR方法从质粒pLenti6/V5-GW/LacZ中获取LacZ全基因,与pEGFP-C1重组后构建真核表达载体pEGFP-C1-LacZ。该重组质粒经PCR和酶切方法鉴定后,在脂质体介导下转染293FT细胞株及鸡胚成纤维细胞,通过荧光观察和组织化学方法检测Egfp和LacZ基因的表达。结果表明,LacZ基因被克隆到pEGFP-C1,成功构建了双报告基因真核表达载体。该重组质粒转染后的细胞呈现绿色荧光,组织化学方法检测到呈蓝色的细胞,表明两个报告基因均能在细胞内正确表达。

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/VS-LacZ plasmid as template. The PCR products of LacZ gene were digested by restriction enzymes of EcoR I and BamH I , 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.