利用抑制性消减杂交（SSH）技术研究不同毒力的鳗弧菌菌株的基因多样性

目的和方法]鳗弧菌是一种嗜盐的革兰氏阴性细菌,也是鱼类弧菌病的主要病原.对斑马鱼的半数致死量研究结果表明,鳗弧菌菌株VIB72具有较高的毒力,而菌株CW1的毒力较低.本文利用抑制性消减杂交(SSH)技术对这两个菌株的遗传差异进行了研究.结果]通过对差减文库筛选,分离到59个对菌株VIB72的阳性克隆,并对这些克隆的DNA序列进行了测定.17个基因片断与其它细菌的已知功能的基因有较高的同源性,其中包括可溶性溶胞壁质转糖基酶、转移蛋白MobA和MobC、转座子IS66、抑制相关蛋白(金属β-内酰胺酶和乙酰转移酶家族)、毒素蛋白(DT-201和alveicin A免疫蛋白)、与OLD家族相似的ATP依赖性核酸内切酶以及SocE和GTP结合蛋白HflX(有高频率的溶原化).这些基因片断有可能是鳗弧菌毒力岛的一部分.其他的基因片断与其它的已知基因没有明显的相关性.结论]这些结果表明,SSH技术成功地鉴定了不同致病性的鳗弧菌菌株的基因差异及潜在的毒力基因.

Genetic diversity between two Vibrio anguillarum strains exhibiting different virulence by suppression subtractive hybridization

Objective] and Methods] Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis in fish.V. anguillarum strain VIB72 was defined as having high virulence whereas strain CW1 was defmed as having low virulence on the basis of their different LD50 values to zebra fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Results] After screening, 59 subtracted library clones were isolated which were specific for strain VIB72, and the DNA sequences of these clones were determined. Seventeen fragments showed high homology to the genes of known functions in other bacteria. This includes soluble lyric murein transglycosylase, mobilization protein (MobA, MobC), transposase (IS66), resistance-related protein (metallo-beta-lactamase and acetyltransferase family), toxin protein (DT-201 and alveicin A immunity protein), ATP-dependent endonuclease of OLD family like protein , SocE and GTP-binding protein HflX (high frequency of lysogenization). These fragments may represent parts of putative pathogenicity islands (PAIs) in V. anguillarum. The remaining fragments showed no significant homology to any known genes.Conclusion] The results indicated that SSH was successful in identifying generic differences and putative virulence genes among different strains of V. anguillarum.