单核细胞增生性李斯特氏菌溶血素基因克隆及序列分析

Cloning and sequence analysis of the hly gene of Listeria Monocytogenes

构建单核细胞增生性李斯特氏菌 (L isteria Monocytogenes,L MO)溶血素基因重组质粒。文章采用 PCR方法扩增出 L MO 0 5 86株溶血素 (Hemolysin,hly)基因 ,将其克隆到 p MD18- T中 ,转化 E.coil TGI。经酶切及 PCR鉴定 ,而后进行测序。 hly基因体外扩增产物大小约为 16 4 6 bp。重组质粒经酶切及 PCR鉴定表明为正确重组子。核苷酸序列鉴定表明 ,其核苷酸序列与国外报道的 L MO F6 789株、L MO F2 36 5株同源性分别为 99.70 %和 99.39%。推导出的氨基酸序列与其相应菌株比较 ,同源性分别为 99.82 %和 98.90 %。在国内首次克隆到 L MO hly全基因 ,为研究hly的功能和探讨 hly蛋白作为特异性诊断靶抗原的研究奠定了基础。

To construct a recombinant plasmid containing hemolysin(hly)gene of Listeria Monocytogenes (LMO).A couple of primers were designed according to the published DNA sequence of LMO hly and were used to amplify the gene for coding the best-characterized determinant of Listeria Monocytogenes (LMO) by polymerase chain reaction (PCR).The purified PCR product was then cloned into pMD18-T vector.The recombinants were screened and identified by restriction analysis and PCR,and the cloned gene was sequenced.The size of amplified hly gene was 1 646 bp.The correct recombinant plasmid was proved by endonuclease analysis and PCR assay.Nucleotide sequence analyses of the hly from the strain indicated that 0586 shared 99.70% and 99.39% identities with F6789(serotype 1/2 b)and F2365(serotype 4 b)respectively and the deduced amino acid sequences were 99.82% and 98.90% correspondingly.The full hly gene was gained firstly in China.This work provided a basis for the further study of the function and diagnosis of hly gene of LMO.