表达绿色荧光蛋白的重组减毒鼠伤寒沙门氏菌的构建与鉴定

Construction and Identification of Recombinant Avirulent Salmonella Typhimurium Expressing Green Fluorescent Protein

应用DNA重组技术构建了表达绿色荧光蛋白 (GFP)的重组减毒鼠伤寒沙门氏菌。在体外稳定试验中 ,重组菌经LB固体及液体培养基连续传 15代后 ,单个菌落和菌液均呈绿色 ;在体内稳定试验中 ,经ICR小鼠连续传代 10次 ,从肝脏、脾脏中分离的细菌经LB固体培养仍为绿色 ,证明构建的重组减毒鼠伤寒沙门氏菌是稳定的。SDS_PAGE结果显示 ,鼠伤寒沙门氏菌表达的GFP分子量约为 2 7kD ,这与预计的分子量大小一致。用免疫剂量的重组细菌接种BALB/C小鼠后 ,观察 1个月未见有异常现象 ,同时剖检后也未见脏器有眼观病变。表达GFP的重组鼠伤寒沙门氏菌的构建为减毒沙门氏菌作为活载体的基因工程疫苗的研制提供一个优良模型 ,为研究沙门氏菌疫苗和沙门氏菌疾病的致病机理提供了一个有力的工具 。

We constructed recombinant avirulent Salmonella typhimurium and confirmed the expression of green fluorescent protein (GFP) by recombinant DNA techniques.The recombinant strains cultured in LB medium through 15 generations still expressed GFP correctly and illuminated green fluorescent,so did the recombinant strains inoculated in ICR rat and then reisolated from its liver and kidney.It showed that the recombinant avirulent Salmonella typhimurium constructed have considerable stability both in vitro and in vivo.The results of SDS-PAGE showed that the 27kD protein of GFP was expressed at pretty high level in Salmonella typhimurium as expected.The recombinant stains were very safe,because no pathology was detected in organs from the BALB/c mice inoculated with the immune dose of recombinant stains.The constructed recombinant avirulent Salmonella typhimurium expressing GFP is a good model for the study of the immune mechanism and development of the recombinant live vaccine with the avirulent Salmonella typhimurium as a live vector.