摘要
[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]This study will be very helpful for identifying the virulent genes,illustrating the pathogenic mechanism and developing the PCR detection kit for virulent strains of V. alginolyticus in the future. [ Methods ] In order to find virulent genes in Vibrio alginolyticus, a subtractive library including 2873 clones was constructed by suppression subtrative hybridization between virulent strain HN08155 and avirulent strain HN08801. [ Results] Two thousand seven hundred and eighty six white clones were amplified by colony PCR. The results of the colony PCR showed that these clones contained the inserted fragments sized from 250 to 1 000 bp. All the PCR products were performed to hybridize to probes of HN08801 with dot southern hybrid- ization. Three hundred and fourty severn colones included the specific fragements of strain HN08155. Then the sequences of the 347 colones were aligned with the published sequence in GenBank. The results of the alignment showed that the library involved 135 sequences of known genes. [ Conclusion]Among these,46 genes were the encoding enzymes genes of several major meta- bolic pathways ; eight genes were for the virulence - relevant genes and 23 genes were for other functions. These virulence - rel- evant genes included serine protease gene prkA, heat shock protein gene dnaJ, chemokine gene cheW, methyl - accepting chem- otaxis protein gene,two- component sensor gene, type IV pilin PilA gene, the biofilm- related associated surface protein gene and outer membrane protein gene. Futhermore, a total of 212 clones contained 166 new unknown genes in the library.
出处
《生物技术》
CAS
CSCD
北大核心
2015年第5期458-462,共5页
Biotechnology
关键词
溶藻弧菌
毒力
抑制性差减杂交
毒力基因
致病机理
Vibrio alginolyticus, virulence, suppression subtrative hybridization, virulent genes, pathogenic mechanism
