乳源致病性金黄葡萄球菌原生质体膜表面蛋白nEBPS的亚细胞定位研究  

Subcellular location of EBPS,a surface protein from protoplasts of pathogenic Staphylococcus aureus found in milk,via an indirect fluorescent-antibody technique

作　　者：吴金花[1,2] 布日额[1,2] 锡林高娃[1,2] 乌日汗[1,2] 薛晓阳[1,2] 刘洋[1,2] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028043 [2]内蒙古民族大学乳源性致病菌研究所

出　　处：《中国病原生物学杂志》2014年第6期509-511,共3页Journal of Pathogen Biology

基　　金：内蒙古自治区科技厅科技创新引导计划项目(No.20111802)

摘　　要：目的克隆牛乳源致病性金黄葡萄球菌nEBPS基因,经工程菌表达得到其编码抗原纯化蛋白,制备多克隆抗体,采用间接荧光抗体法鉴定nEBPS蛋白。方法设计1对克隆引物,PCR扩增牛乳腺炎金黄葡萄球菌内蒙古分离株EBPS基因序列,构建重组表达质粒,转化工程菌,表达膜表面亚单位蛋白nEBPS,纯化后免疫家兔制备抗nEBPS蛋白抗体,采用间接荧光抗体法对乳源致病性金黄葡萄球菌原生质体膜表面蛋白nEBPS进行亚细胞定位。结果在含25μg/ml Amp、0.015g/ml NaCl的LB液体培养基中成功培养制备了金黄葡萄球菌原生质体。对原生质体细胞(表面)和利用0.02g/ml NaCl高渗溶液裂解原生质体细胞离心获得的细胞膜残片进行间接荧光抗体试验,均出现特异荧光。结论间接荧光抗体试验鉴定nEBPS蛋白为膜表面蛋白,可作为乳及乳制品金黄葡萄球菌检测和制备免疫制剂的靶抗原。Objectives To clime the nEBPS gene of pathogenic Staphylococcus aureus from milk and to identify nEBPS proteins as membrane proteins via an indirect fluorescent-antibody technique with polyelonal antibodies against nEBPS protein. Methods A pair of primers was designed for amplification of the nEBPS gene sequence and a recombinant plasmid was constructed. The nEBPS protein was expressed and purified, and then rabbits were immunized to prepare anti bodies against that protein, nEBPS protein was verified to be a membrane surface protein by indirect fluorescent-antibody identification, Results Cells of S. aureus protoplasts were successfully prepared in LB liquid medium containing 25 μg/ ml Amp and 0. 015 g/ml of NaCI. Indirect fluorescent-antibody identification revealed nEBPS protein on the surface of the cell membranes of protoplasts and membrane fragments tested positive for the protein. Conclusion nEBPS protein is a membrane surface protein and can be used as to detect Staphylococcus aureus in milk and milk products, nEBPS protein can also serve as a target antigen to prepare immunizing agents.

关 键 词：nEBPS基因 原生质体 间接荧光抗体 

分 类 号：R378.11[医药卫生—病原生物学]