肠出血性大肠杆菌O157:H7的紧密黏附素基因eae的测序及生物信息学分析

目的 克隆编码肠出血型大肠杆菌O157:H7的紧密黏附素eae基因,并对其进行测序及生物信息学分析,为研究EHEC与宿主细胞相互作用奠定基础.方法 利用PCR技术扩增eae基因,经过纯化,将其定向插入克隆载体PMD19-T simple vector并进行测序,应用生物信息学软件分析其生物学特性.结果 用PCR方法扩增eae基因,大小为2802 bp,编码934个氨基酸,用DNAstar5.0软件分析紧密黏附素(Intimin)蛋白的生物活性,在202～210、510～520、716～735个氨基酸残基的肽链上,显示该蛋白具有良好的亲水性,在175～220、300～315、550～610、710～780、825～880个氨基酸残基的肽链上具有良好的柔韧性,在220～300、615～710个氨基酸残基的肽链上,显示该蛋白具有良好的抗原性.结论 本研究用PCR法扩增出了O157:H7的eae基因,成功构建了肠出血性大肠杆菌O157:H7紧密黏附素eae基因的重组质粒,并分析了黏附素蛋白的亲水性、柔韧性、抗原性,为进一步研究大肠杆菌O157:H7黏附素的致病机制奠定了基础,为下一步表达出Intimin蛋白,进一步研究该蛋白与宿主细胞的粘附、免疫原性、抗原抗体反应提供了理论依据.

Bioinformatics Analysis of the Intimin eae Gene of Enterohemorrhagic E.coli(EHEC)O157:H7

Objective To clone and sequence the eae gene of EHEC, and to provide information for the future analysis of the interaction between EHEC and the host cells. Method eae gene from EHEC was amplified by PCR. The purified target fragment was then cloned into plasmid PMD19-T simple vector. The cloned fragment was confirmed by sequencing. The biological properties of the gene were analyzed by DNAstar 5.0. Result A fragment of eae gene containing 2802 bp, coding for a polypeptide of 934 amino acids, was obtained from the genomic DNA of an EHEC Guangzhou strain. Sequence analysis predicted that the encoded peptide possessed antigenic determinants at the amino acid residues between 220-300 and 615-710. The peptide also possessed hydrophilic regions at the amino acid residues between 202-210, 510-520 and 716-735; and the flexible regions at the residues between 175-200, 300-315,550-610 and 710-780. Conclusion The eae gene fragment from EHEC was cloned into PMD19-T plasmid. Information obtained from sequence analysis will be used for the future study of the disease caused by EHEC.