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[目的]通过构建基因文库为溶藻弧菌毒力基因的鉴定、致病机理的阐明奠定基础。[方法]应用抑制性差减杂交技术(suppression subtrative hybridization,SSH),以毒力菌株HN08155为测试子(Tester),构建了溶藻弧菌毒力菌株HN08155的SSH差减文库。[结果]文库共含有2 873个克隆;通过菌落PCR鉴定,2 786个克隆能扩增出250~1 000bp大小的目标片段;通过Southern斑点杂交,确定毒力菌株特异基因的阳性克隆347个,经测序和同源性比对分析,其中135个克隆的序列含有已知的77个基因,其中46个基因主要为代谢途径的多种酶类的编码基因,23个基因为其它基因,8个为毒力相关功能基因。[结论]这些毒力相关功能基因包括丝氨酸蛋白酶基因prk A、热休克蛋白基因dna J、趋化因子基因che W、甲基受体趋化蛋白(methyl-accepting chemotaxis protein)基因、双组分感应子(two-component sensor)基因、Ⅳ型菌毛(typeⅣpilin Pil A)基因、生物膜相关的表面蛋白(biofilm-associated surface protein)基因和外膜蛋白(outer membrane protein)基因;该文库中还含有166个未知新基因,占文库中总基因数的68.44%,溶藻弧菌的毒力基因很可能存在于这个文库中的未知新基因中。
[Objective] The research aimed to establish the gene library for the identification of virulence genes of Vibrio alginolyticus and elucidation of the pathogenesis. [Method] SSH subtractive library of V. alginolyticus virulent strain HN08155 was constructed by using suppression subtractive hybridization (SSH) and virulent strain HN08155 as tester. [Result] A total of 2 873 clones were obtained in the library. A total of 2 786 clones amplified 250 to 1 000 bp by colony PCR. A total of 347 positive clones of virulent strain-specific genes were identified by Southern blotting. Sequence analysis and homology analysis showed that 135 of the cloned sequences contained 77 known genes, of which 46 genes were mainly coding genes of various enzymes in metabolic pathway, 23 genes were other genes, and 8 Virulence-related functional genes. [Conclusion] These virulence related functional genes include serine protease gene prk A, heat shock protein gene dna J, chemokine gene che W, methyl-accepting chemotaxis protein gene, two-component induction A two-component sensor gene, a type IV pilin Pil A gene, a biofilm-associated surface protein gene and an outer membrane protein gene; this library also contains 166 unknown new genes, accounting for 68.44% of the total number of genes in the library, Vibrio alginolyticus virulence genes are likely to exist in this library of unknown new genes.