EHEC O157：H7紧密黏附素C300与LTB融合基因克隆与序列分析

目的构建肠出血性大肠埃希菌（EHEC）O157：H7紧密黏附素C-端免疫保护片段与肠黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位（LTB）的融合基因克隆载体,为进一步研究EHEC O157：H7重组疫苗奠定了基础.方法设计引物采用PCR技术从EHEC O157：H7基因组中扩增Intimin C300的编码基因,从肠产毒性大肠埃希菌基因组中扩增LTB的编码基因,分别构建克隆载体pUC18-C300和pUC18-LTB,采用酶切位点相连技术,构建pUC19-C300-LTB克隆载体,并测序.结果从EHEC O157：H7基因组中扩增出了900 bp的目的片段,从肠产毒素大肠埃希菌基因组扩增出了275 bp的目的片段,分别插入载体pUC18和pUC19,经酶切及测序鉴定与目的序列一致.结论克隆出EHEC O157：H7的紧密黏附素C-端免疫保护片段与不耐热肠毒素B亚单位基因,并成功构建融合基因克隆载体,测序正确.     

Phylogenetic background of attaching and effacing <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from animals

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.     

Preface

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the α1, α2, β, γ1, γ2/θ, κ, ɛ, ζ, and ι intimin variants.