肠出血性大肠杆菌O157∶H7 eae基因原核表达及间接ELISA的初步建立

Prokaryotic Expression of Enterohemorrhagic Escherichia coli O157:H7 eae Gene and Preliminary Establishment of an Indireet ELISA

对肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)O157∶H7 LEE毒力岛上的eae基因进行克隆与原核表达,以表达产物紧密素(intimin)为包被抗原,初步建立O157∶H7血清抗体间接ELISA。结果表明,抗原最佳包被浓度为0.88μg/mL,血清最佳稀释倍数为200;与鸡大肠杆菌其他血清型O1、O2、O11、O18、O35、O45、O78、O86、O88、O147、鸡白痢以及新城疫等血清均无交叉反应,该方法特异性较好;血清稀释倍数为1 600倍时仍能检测到O157∶H7 intimin特异性抗体,该方法灵敏度较高。

An indirect enzyme-linked immunorbent assay （ELISA） was developed to facilitate early detection of Enterohemorrhagic Escherichia coli（EHEC）O157∶H7.The antigen was a recombinant protein intimin,which was the product of the conserved eae gene in LEE of EHEC O157∶H7.The coated material was recombinant protein intimin after purified.The optimal concentration of antigen was 0.88 μg/mL.The best test serum was used after dilution 200.The intimin-based ELISA successfully detected intimin antibody of EHEC O157∶H7.There were no cross-reaction with Escherichia coli O1、O2、O11、O18、O35、O45、O78、O86、O88、O147、Pullorum disease、Newcastle Disease.With this method,the intimin antibody could be still detected from serum used after dilution 1 600,indicating that the method was sensitive.