原核真核双表达加强型绿色荧光蛋白质粒的构建

CHARACTERIZATION OF A PROKARYOTIC-EUKARYOTIC DOUBLE EXPRESSION PLASMID EXPRESSING ENHANCED GREEN FLUORESCENCE PROTEIN

将原核启动子 Ptrc和加强型绿色荧光蛋白 ( EGFP)基因从 EGFP原核表达质粒 p YA3 3 3 4-EGFP上切下 ,然后将其插入至真核表达质粒 p VAX1真核启动子 Pcmv的下游 ,构建成具有真核和原核启动子的双表达杂合质粒 p VAXD-EGFP,其长度为 3 82 3 bp。将 p VAXD-EGFP分别转化鼠伤寒沙门氏菌 X45 5 0和转染 COS-7细胞 ,应用流式细胞术、可见光谱扫描、SDS-PAGE测定 EGFP原核表达 ;应用荧光显微镜观察 EGFP在 COS-7细胞内的表达。重组鼠伤寒沙门氏菌 X45 5 0 ( p VAXD-EGFP)表达 EGFP的量与仅以原核方式表达 EGFP的 X45 5 0 ( p YA3 3 3 4-EGFP)相当 ;将质粒p VAXD-EGFP转染 COS-7细胞 ,EGFP可在 COS-7细胞核和胞浆表达 ,在荧光显微镜下发出强烈荧光。结果表明 :不仅成功地构建原核、真核表达集中于同一质粒的新型质粒 p VAXD-EGFP,而且原核、真核目的蛋白表达量达到很高水平 。

Prokaryotic promoter Ptrc and enhanced green fluorescence protein (EGFP) gene was excised from EGFP prokaryotic expression plasmid pYA3334 EGFP and inserted into the downstream of eukaryotic expression promoter Pcmv in DNA vaccine vector pVAX1. This new double expression plasmid was named as pVAXD EGFP and consequently transformed into Salmonella typhimurium X4550 and transfected into COS 7 cell. The EGFP expression in X4550 (pVAXD EGFP) was detected by flow cytometer, visible spectrum scanning and SDS PAGE and its expression in COS 7 cell was monitored by fluorescence microscope. EGFP was expressed both in X4550 and COS 7 cell and its expression level was comparable with corresponding recombinant S.typhimurium X4550 (pYA3334 EGFP) and EGFP eukaryotic expression plasmid pVAX1 EGFP. A prokaryotic eukaryotic double expression plasmid was successfully constructed and it has potential applications in designing new recombinant Salmonella vaccines.