绿色荧光蛋白标记穿梭表达载体构建及重组乳酸菌制备.pdfVIP

( ) 吉林农业大学学报 　2009 ,31 3 :325～329 http :/ / x uebaoj lau educn J ournal of J ilin A gricultural University Email :j lndxb @vip sinacom 绿色荧光蛋白标记穿梭表达载体构建及重组乳 酸菌制备 陈晓雷, 杨桂连, 王春凤 吉林农业大学动物科技学院 ,长春 130118 摘 　要 : 以红霉素耐药性基因/ thy A 基因双筛选压力大肠杆菌 —乳酸杆菌穿梭表达载体 pW425et 为基本骨 ( ) 架 ,将绿色荧光蛋白 GFP 基因插入该载体 ,构建 GFP 标记的穿梭表达载体 pW425etGFP 。通过连续的荧光强 度检测 ,验证 gf p 基因能否在重组菌中得到稳定表达 。重组表达质粒 pW425etGFP 酶切和 PCR 鉴定结果与预 期片段大小相符 ,SDS - PAGE 显示 27 kD 的绿色荧光蛋白获得表达 ,在蓝光的激发下 ,可见明显的绿色荧光 , 且荧光稳定性强 ,连续传代 30 次仍可见荧光 。结果表明已成功构建了 GFP 标记穿梭表达载体 pW425etGFP , 为乳酸菌作为益生生理菌载体进行体内定植规律研究和食品级 、疫苗级乳酸菌产品的开发奠定了基础 。 关键词 : 绿色荧光蛋白; 穿梭表达载体 ; 大肠杆菌 ; 乳酸菌 ( ) 中图分类号 : S8526 　　　文献标识码 : A 　　　文章编号 : 2009 Construction of GFP Labeling Shuttle Expression Vector and Prepara tion of Restitution L actobacillus CHEN Xiaolei , YAN G Guilian , WAN G Chunf eng College of A nimal Science and Technology , J ilin A gricultural University , Changchun 130118 , China Ab stract : GFP labeling shuttle expression vector pW425etGFP was constructed by inserting green fluo ( ) rescent protein GFP based on erythromycin drug resistance gene / thy A gene diplbolting pressure shuttle expression vector pW425et The expression stability of GFP was estimated by consecutive fluorescence in tensity detection Results of enzyme digested and PCR identify of pW425etGFP were consistent with those expected SDS - PA GE showed that 27 kD GFP had been expressed Green fluorescent which could be observed in the blue light were of higher stability after 30 serial passages Results showed t