溶藻弧菌溶血活性检测及溶血素基因、启动子区功能

摘要

[目的]研究溶藻弧菌的溶血现象,溶血素基因vah的分布及vah基因、vah启动子区对溶藻弧菌溶血活性的贡献.[方法]对46株分离自华南沿海水生动物体内和海水的溶藻弧菌环境株及溶藻弧菌标准株1.1587进行溶血实验；比较具有溶血活性的溶藻弧菌野生株ZJ051、vah基因大肠杆菌BL21重组表达株、vah缺失突变株和基因回补株间溶血能力的差异；检测vah基因在溶藻弧菌中的分布,比较溶血株与非溶血株vah基因及上游启动子区的序列差异.[结果]47.8％的溶藻弧菌菌株产生溶血活性,因此溶血现象普遍存在于溶藻弧菌环境株中；vah基因的表达产物具有溶血活性,vah基因缺失突变株不具有溶血活性,而vah基因回补株恢复溶血活性.vah基因普遍存在于溶藻弧菌中,且基因序列非常相似,氨基酸序列完全相同,然而不同菌株的启动子区第188-190碱基位点存在差异.[结论]溶藻弧菌vah基因是造成溶藻弧菌溶血的直接原因,但溶藻弧菌溶血能力的差异并非是由vah基因本身差异决定,极有可能与启动子区第188-190碱基位点相关.%[Objective] We studied the hemolytic activity of Vibrio alginolyticus strains,and the distribution of a hemolysin gene vah in these strains;we analyzed the functions of vah gene and the related promoter,and assessed their contributions to the hemolytic activity.[Methods] Hemolytic test was performed using 47 V.alginolyticus strains,including one type strain 1.1587 and 46 environmental strains isolated from seawater and various marine animals.The difference of hemolytic ability was compared among a wild V.alginolyticus strain ZJ051 that showed hemolytic activity,a recombinant Escherichia coli BL21 aiming at a predicted vah gene,a vah-deletion mutant,and a vah-recovery strain.The distribution of vah gene was further detected in V.alginolyticus followed by the sequence analysis of vah genes and their promoters among the hemolytic and non-hemolytic strains.[Results] Among the V.alginolyticus strains 47.8％ showed hemolytic activities,and thus hemolysis was ubiquitous in environmental V.alginolyticus strains.Hemolysis was observed in the recombinant Escherichia coli BL21 and the vah-recovery strain but not in the vah-deletion mutant.The vah gene widely distributed in V.alginolyticus.The vah sequences of V.alginolyticus strains were highly similar,and they shared identical amino acid sequences.Through the vah promoter comparison between the hemolytic and non-hemolytic strains,a difference in 188-190 base sites of the promoters was discovered.[Conclusion] Hemolysis is directly caused by the vah gene,and the discrepancy of hemolytic ability among various V.alginolyticus strains has no relation with the difference of vah sequences,which is likely determined by the diversity in 188-190 base sites of vah promoters.