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目的 利用抑制消减杂交技术 (SSH) ,比较致病与非致病钩端螺旋体 (简称钩体 )之间基因组差异 ,筛选致病钩体特有的基因片段。方法 以赖型 0 1 7株为tester,非致病性patocⅠ株为driv er,选择合适的四碱基内切酶将基因组酶切 ,连接特殊设计的adaptor进行消减杂交和PCR ,得到消减混合物 ,与T/A克隆载体连接 ,转化JM1 0 9建立差异消减文库 ,经PCR和Southernblot筛选鉴定阳性克隆 ,进而对部分片段进行测序和同源分析。结果 AluⅠ适于将钩体酶切成用于SSH技术的小片段 ;经SSH筛选鉴定得到 3 0个片段大小为 2 0 0～ 1 3 0 0bp的阳性克隆 ,部分已测序的片段中 1个与硫胺素合成蛋白高度同源 ,4个与GenBank无明显同源性 ,可能为赖型致病钩体所特有而非致病钩体缺失的基因片段 ,已被GenBank收录 ,收录号分别为AF3 0 0 873～ 3 0 0 877。结论 SSH是一种有效、灵敏、高特异的比较分析基因组差异的方法 ,对钩端螺旋体致病基因的筛选、基因组分化、分子遗传研究等具有重要意义。
Objective To compare the genomic differences between pathogenic and non-pathogenic Leptospira (SSH) by SSH and screen out the unique gene fragments of pathogenic Leptospira. Methods The strain 017 was used as tester, the non-pathogenic patocⅠ strain was driv er. The appropriate four-base endonuclease was used to digest the genome and ligated to a specially designed adapter for subtractive hybridization and PCR. Was ligated with T / A cloning vector and transformed into JM109 to establish differential subtractive library. The positive clones were screened by PCR and Southern blotting, and the partial fragments were sequenced and homologous analyzed. Results AluⅠ was suitable for digestion of the snail into small fragments for SSH. 30 SSH positive clones were screened by SSH. One of the partially sequenced fragments was Thiamine synthesis protein highly homologous, four with no significant homology with GenBank, may be dependent on the leishmania specificity of non-pathogenic Leptospira gene fragments, has been GenBank, accession numbers were AF3 0 0 873 ~ 3 0 0 877. Conclusion SSH is an effective, sensitive and highly specific method for comparative analysis of genomic differences. It is of great significance for the screening of Leptospira virulence genes, genomic differentiation and molecular genetic studies.