利用抑制消减杂交技术研究钩端螺旋体致病相关基因

Isolation of pathogenicity related genes of Leptospira serovar lai by suppression subtractive hybridization (SSH)

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 isolate the pathogenicity related genes of Leptospira serovar lai. Methods Using suppression subtractive hybridization (SSH) technique, we compared the genomic difference between pathogenic Leptospira serovar lai (tester) and nonpathogenic Leptospira serovar patocⅠ(driver). Two subtractive hybridization and PCR were performed. The mixture of subtracted DNA fragments were cloned and transformed to establish the differential subtraction library. The positive clones were identified by PCR and Southern blot, then sequenced and searched homologically. Results Leptospira DNA could be digested by AluⅠfor SSH. 30 positive clones were screened by SSH. These 200-1?300bp fragments were enriched in Leptospira serovar lai genomic sequences that are absent from the nonpathogenic Leptospira . Parts of these fragments were sequenced and searched homologically in GenBank, 1 of them was high homology with thimaine biosynthesis protein, 4 of them were identified as novel gene fragments showing no significant homology to any known sequences, which deposited respectively in GenBank(AF300873-300877). Conclusion SSH is an effective, sensitive method for comparing genomic differences and isolating pathogenicity related genes.