尿路致病性大肠杆菌溶血素A基因的克隆和序列分析

Cloning and sequence analysis of the hlya gene of Uropathogenic Escherichia coli

目的构建尿路致病性大肠杆菌(Uropathogenic Escherichia coli,UPEC)溶血素A(hemolysin A,HLYA)hlya基因重组质粒。方法根据已知GenBank中的hlya基因序列,设计合成一对引物,用PCR方法从尿路致病性大肠杆菌溶血素A基因组DNA中扩增编码hlya的基因片段,克隆至pUC18质粒,转化大肠杆菌DH5a感受态细胞,经酶切及PCR鉴定,而后进行测序。结果hlya基因体外扩增产物大小约744bp,重组质粒经酶切及PCR鉴定表明获得正确重组子,测序结果与已知序列基本吻合。结论在国内首次克隆了尿路致病性大肠杆菌hlya基因,为研究hlya的功能和探讨hlya作为特异性诊断靶抗原的研究奠定了基础。

Objective： To construct a recombinant plasmid containing hlya gene of Uropathogenic Escherichia coli （upec）. Methods ： A couple of primers were designed for PCR according to the known sequence of hlya gene. The hlya gene obtained by amplification from genomic DNA of Uropathogenic Escherichia coti （upec） by PCR technique was cloned into plasmid pUC18 directionally ,the constructed recombinant plasmid was transferred into E. coliDH5a. The transformants were screened and identified by restriction analysis and PCR. Then the cloned genes were sequenced. Results： The size of amplified hlya gene was 744bp. The correct recombinant plasmid pucl8-hlya was isolated and confirmed by restruction analysis and PCR. DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence. Conclusion： The hlya gene can be successfully amplified and cloned into plasmid pUC18. The study provides the basis for the further study of the function and diagnosis of hlya gene of Uropathogenic Escheriehia coli（upec）