金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

Selection and Application of DNA Aptamers Specifically to Staphylococcal aureus Enterotoxin B

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。

Objective：The systematic evolution of ligands by exponential enrichment （SELEX） was applied for screening single strand DNA（ssDNA） aptamers which specifically bind to Staphylococcal aureus enterotoxin B（SEB）,and was used in clinical detection.Methods：A 96 nt random ssDNA library was designed and synthesized in vitro,and the dynabeads carboxylic acid was as medium in the SELEX,the ssDNA aptamers specifically binding to SEB were obtained through PCR amplification and selection.The aptamer exhibited binding ability to protein target was identified by fluorescent assay.The specificity and affinity were tested in an enzyme-linked oligonucleotide assay.Results：After 13 rounds of selection,the binding ability of the aptamers was from 1.1% to 39.8%,36 times more than the first round.It had been shown that aptamers bind only to SEB but not to SPA,and it could initially discriminate the blood serum of patient from control.Conclusion：The ssDNA aptamers （A11） specifically binding to SEB has been selected by using SELEX,providing a foundation for investigation and therapy infection of caused by S.aureus.