副溶血弧菌O抗原基因簇中庚糖基转移酶Ⅱ基因缺失株的构建及其功能

Gene deletion and functional analysis of the heptyl glycosyltransferaseⅡ (waaF) gene in Vibrio parahemolyticus O-antigen cluster

摘要:

[目的] 构建副溶血弧菌庚糖基转移酶Ⅱ基因(waaF)的缺失株,探究waaF基因在副溶血弧菌O抗原合成中的作用。[方法] 本研究以副溶血弧菌临床分离株为研究对象,利用甲壳素介导的转化技术构建临床分离株的waaF基因缺失株;分别对野生株、缺失株的生长曲线、菌体形态和血清型进行了测定;利用大肠杆菌S17λpir菌株与副溶血弧菌结合转移的方法,分别构建O3、O5和O10来源的waaF基因的回补株,通过血清型测定,验证同源waaF基因的功能。[结果] 成功构建了waaF基因缺失株,基因缺失株生长正常,其生长曲线、菌体形态同野生菌株基本一致,基因缺失株同O抗血清不发生凝集反应,O抗原特性消失。回补实验显示,O3和O5来源waaF基因的回补株能恢复原有O抗原特性,O10来源waaF基因的回补株则不能恢复基因缺失株的O抗原特性。[结论] waaF基因同O抗原的合成相关,是O抗原合成的关键基因,不同O抗原副溶血弧菌中waaF基因功能存在差异。

关键词:副溶血弧菌;O抗原;waaF基因;功能分析

Abstract:

[Objective] To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. [Methods] The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. [Results] The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. [Conclusion] The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Key words:Vibrio parahemolyticus;O-antigen;waaF gene;functional analysis