不同生长阶段大肠杆菌O157:H7适配体的筛选及鉴定
首发时间:2018-03-27
摘要:大肠杆菌O157 : H7(Escherichia coli O157:H7, E.coli O157:H7)是肠出血性大肠杆菌(Enteroheamorrhagic Escherichia coli, EHEC)主要的致病菌株,其致病性主要是由其分泌的志贺毒素及其变体而引起,毒性极强,对公众的健康造成了巨大的威胁。因此,实现对大肠杆菌O157:H7准确快速的检测至关重要。在本研究中,考虑到志贺毒素在大肠杆菌O157:H7的不同生长时期均有存在,而且不同生长时期的细菌外膜结构可能存在差异,因此本实验采取不同生长时期的大肠杆菌O157:H7作为靶标,利用Cell-SELEX技术,通过十四轮的筛选(包括三轮反筛),成功获得能与不同生长时期大肠杆菌O157 : H7特异性结合的寡核苷酸适配体。接着通过克隆测序,获得32条寡核苷酸序列并对其进行同源性和二级结构分析,挑选出9条序列进行亲和性和特异性分析。最后利用流式细胞术分析得出序列Apt-5具有良好的亲和力和特异性,解离常数Kd值为9.04±2.08 nmol/L。
关键词: 大肠杆菌O157:H7 适配体 SELEX 流式细胞术
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Selection and Identification of an ssDNA Aptamer Targeted to different stages of E.coli O157:H7
Abstract:Enterohemorrhagic Escherichia coli O157:H7 (E.coli O157:H7) is known as an important food-borne pathogens related to public health. It can cause haemolytic uraemic syndrome (HUS) mainly by secretion of Shiga toxins and variants. It poses a great threat to public health. Therefore, it is quite important to develop a rapid, sensitive and cost-effective analysis method for E. coli O157:H7. In this study, considering that Shiga toxin exists in different growth stages of E. coli O157:H7, and there may be differences in the outer membrane structure of bacteria at different growth stages, E. coli O157:H7 at different growth stages was used as the target. Aptamers which could bind to different stages of E.coli O157:H7 with high affinity and specificity was obtained by Whole cell-selex method through fourteen selection rounds including three counter-selection rounds. To analyze affinity and specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the optimal aptamer that recognize different stages of E.coli O157:H7 specifically with the Kd value of 9.04±2.80 nM.
Keywords: E.coli O157:H7 aptamer SELEX flow cytometry
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