产A型肠毒素金黄色葡萄球菌的筛选及其重组表达  

Cloning and expression of the type A staphylococcal enterotoxin gene from wild strain of Staphylococcus aureus

作　　者：吴双[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 [2]深圳大学生命科学学院

出　　处：《中国人兽共患病学报》2008年第5期451-454,共4页Chinese Journal of Zoonoses

基　　金：深圳市农业综合开发基金(No.2004-33)

摘　　要：目的分离产A型肠毒素金黄色葡萄球菌标准株,克隆和表达金黄色葡萄球菌肠毒素A(SEA),研究其生物学特征。方法应用PCR扩增SEA基因片段,与克隆载体pGEMT-easy连接,亚克隆到表达载体pET-28a中,转化大肠杆菌BL21(DE3),经诱导表达和纯化重组蛋白,用重组蛋白免疫小鼠获得抗血清,进行重组蛋白免疫原性分析。结果成功克隆SEA全长基因,经DNA序列分析证实与GenBank收录序列具有100%的同源性。表达蛋白相对分子质量为31000,以包涵体形式存在。纯化、复性后获得有活性的rSEA蛋白,rSEA蛋白能够识别天然的SEA。结论SEA基因成功克隆到表达质粒内并表达,rSEA具备抗原性,为制备单抗、诊断试剂及金黄色葡萄球菌致病机制研究奠定了基础。To clone,identify and express the staphylococcal enterotoxin A（SEA）gene from wild strain of Staphylococcus a ureus, the SEA gene fragment was amplified by PCR,ligated to the cloning vector pGEM T-easy ,cloned to the expression vector pET-28a and then transformed to E. coli BL21（DE3）. The recombinant SEA （rSEA）thus obtained was expressed after induction with IPTG and then purified. Anti-rSEA serum was prepared by the immunization of mice with rSEA,and this antiserum was used in the analysis of immunogenicity of rSEA as revealed by the Western blotting. It was demonstrated that the totallength of the SEA gene was cloned successively,and it had been proved to have 100% homology with the published sequence in GenBank through the DNA sequence analysis. The protein with a relative molecular mass of 31000 was expressed as inclusion bodies after purification and renaturation,and the antiserum against rSEA could recognize the native SEA. It is evident that the rSEA obtained shows excellent immnogencity, thus paving the way for development of monoclonal antibodies and diagnostic reagents as well as for the study on the pathogenicity of Staphlococcus aureus infections.

关 键 词：金黄色葡萄球菌A型肠毒素 重组表达 蛋白纯化 复性 

分 类 号：R378.1[医药卫生—病原生物学]