产肠毒素D金黄色葡萄球菌的筛选及其基因克隆和蛋白表达  

Isolation of Type D Enterotoxigenic Staphylococcus aureus and Cloning and Expression of Enterotoxin D Gene

作　　者：甄茵[1] 张仁利[1] 耿艺介[1] 高世同[1] 胡章立[2] 

机构地区：[1]深圳市疾病预防控制中心,深圳518020 [2]深圳大学生命科学学院,深圳518060

出　　处：《热带医学杂志》2008年第1期23-27,共5页Journal of Tropical Medicine

基　　金：深圳市农业综合开发基金(No.2004-33)

摘　　要：目的克隆金黄色葡萄球菌肠毒素D基因(entD),表达和纯化其重组蛋白(rSED),以进一步制备抗rSED抗体,研制SED金标免疫快速检测试纸条。方法利用PCR技术,从野生型金黄色葡萄球菌中筛选产肠毒素D金黄色葡萄球菌标准株,扩增entD基因,克隆至pGEM-T Easy载体,亚克隆至原核表达载体pET28a,重组质粒转化到大肠杆菌BL21(DE3),IPTG诱导表达蛋白,Ni-NTA亲和层析纯化蛋白,免疫印迹检测抗原活性。结果分离获得1株产肠毒素D金黄色葡葡球菌标准株,成功克隆entD序列,测序表明该基因共684 bp,与其他entD序列(GenBank登陆号:M28521)具有99%的同源性。rSED蛋白在大肠杆菌中得到高效的可溶性表达。免疫印迹结果表明,rSED蛋白能被抗天然SED抗体识别。结论通过基因重组技术制备的重组SED蛋白具有良好的免疫反应性。Objective To clone staphylococcal enterotoxin D gene （entD）, express and purify staphylococcal enterotoxin D （rSED） recombinant protein for the preparation of anti-rSED antibody and development of a colloidal gold immunochromatography test strip for rapid detecting SED. Methods Firstly, PCR was carried out to screen SED producing strain of Staphylococcus aureus from wild type S. aureus. Then entD gene was isolated and cloned into pGEMT Easy vector. The expression vector pET28a-SED was constructed to express rSED protein in Escherichia coli BL21 （DE3）. The rSED protein was purified by Ni-NTA column and its immunological activity was analyzed by western blot. Results One type D enterotoxigenic S. aureus was isolated and entD gene was successfully cloned. The sequencing result showed that the cloned entD gene contains 684 nucleotides coding for 228 amino acids,and was 99% identical to the sequence reported in Genbank （Accession number： M28521 ）. Soluble rSED protein could be expressed effectively in E.coli BL21 （DE3）. Western blot result indicated that rSED protein reacted with the rabbit anti-native SED polyclonal antibody. Conclusion The rSED protein has a good immunological activity and may be able to use for diagnosis.

关 键 词：金黄色葡萄球菌肠毒素D 重组SED蛋白 

分 类 号：R378.11[医药卫生—病原生物学]