表达绿色荧光蛋白重组单核细胞增多性李斯特菌的构建(英文)  

Construction of the recombinantListeria monocytogenes mutant expressing green fluorescent protein

作　　者：柯春林[1] 宋厚辉[2] 江玲丽[1] 张桂芝[1] 方维焕[1] 

机构地区：[1]浙江大学动物预防医学研究所,浙江省动物预防医学重点实验室,浙江杭州310029 [2]中国科学院微生物研究所,北京100080

出　　处：《浙江大学学报（农业与生命科学版）》2005年第5期638-644,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：SponsoredbytheNationalScienceFoundationofChina(ProjectNo.30270073)

摘　　要：将编码绿色荧光蛋白(GFP)的基因融合到单核细胞增多性李斯特菌actA基因启动子和信号肽下游,应用同源重组技术构建了含gfp的重组李斯特菌突变株.PCR扩增及其产物的酶切鉴定均证实目的基因gfp融合到李斯特菌基因组中.荧光显微镜下可见重组菌体发绿色荧光.SDS-PAGE电泳和免疫印迹结果显示重组李斯特菌表达的GFP分子量约为28 kD,与预计的分子量大小一致.重组菌对鸡胚和小鼠的毒力以及对体外培养细胞的侵袭力均低于野生型李斯特菌.该重组菌可用于研究李斯特菌的致病机理和食品加工过程中微生物污染控制的指示性细菌.A recombinant Listeria monocyogenes mutant expressing green fluorescent protein （GFP） gene was constructed by homologous recombination. The target gene gfp was fused to the actA gene fragment downstream of its promoter and signal sequence. PCR amplification and restriction digestion of the PCR product confirmed the insertional fusion. The reporter gene was expressed as shown by fluorescent microscopy and SDS-PAGE/western blotting. The recombinant strain exhibited reduced invasivenes.s to the in vitro cultured cells as well as decreased virulence to the chicken embryos and mice. The strain has potential applications in microbe-host interaction studies in the medical or veterinary medical fields and in food microbiological studies.

关 键 词：单核细胞增多性李斯特菌 同源重组 绿色荧光蛋白 外源基因表达 

分 类 号：S852.11[农业科学—基础兽医学]