绿色荧光蛋白标记穿梭表达载体构建及重组乳酸菌制备  

Construction of GFP Labeling Shuttle Expression Vector and Preparation of Restitution Lactobacillus

作　　者：陈晓雷[1] 杨桂连[1] 王春凤[1] 

机构地区：[1]吉林农业大学动物科技学院,长春130118

出　　处：《吉林农业大学学报》2009年第3期325-329,共5页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(30671573);国家高技术研究发展计划项目(2006AA10A205;2007AA10Z322)

摘　　要：以红霉素耐药性基因/thyA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pW425et-GFP。通过连续的荧光强度检测,验证gfp基因能否在重组菌中得到稳定表达。重组表达质粒pW425et-GFP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pW425et-GFP,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。GFP labeling shuttle expression vector pW425et-GFP was constructed by inserting green fluorescent protein(GFP)based on erythromycin drug resistance gene /thyA gene dipl-bolting pressure shuttle expression vector pW425et.The expression stability of GFP was estimated by consecutive fluorescence intensity detection.Results of enzyme digested and PCR identify of pW425et-GFP were consistent with those expected.SDS-PAGE 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 that GFP labeling shuttle expression vector has been constructed successfully.It establishes the foundation for the research of field planting axiom in vivo as well as the development of the food-grade and the vaccine-grade lactic acid bacteria production of lactobacillus as a physiobacterium vector.

关 键 词：绿色荧光蛋白 穿梭表达载体 大肠杆菌 乳酸菌 

分 类 号：S852.6[农业科学—基础兽医学]