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【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。
【Objective】 To construct the deletion mutant of waaF gene of Vibrio parahaemolyticus, and to explore the role of waaF gene in the synthesis of V. parahaemolyticus O antigen. 【Method】 In this study, Vibrio parahaemolyticus clinical isolates were selected as the research objects, and chitin-mediated transformation was used to construct the waaF gene-deleted strains of clinical isolates. The growth curves, morphology and The serotypes were determined. The complement of waaF gene from O3, O5 and O10 was constructed by the combination of Escherichia coli strain S17λpir and Vibrio parahaemolyticus. The function of the homologous waaF gene was tested by serotyping. 【Result】 The waaF gene deletion mutant was successfully constructed. The gene deletion mutant grew normally. The growth curve and morphology of the mutant strain were basically the same as those of the wild strain. The deletion of the gene deletion strain and the O antiserum did not occur, and the O antigen disappeared. Replenishment experiments showed that O3 and O5 replenishment of waaF gene restore the original O antigen characteristics, O10 source waaF gene recapitulation strain can not restore gene deletion strain O antigen characteristics. 【Conclusion】 The waaF gene is related to the synthesis of O antigen and is the key gene for the synthesis of O antigen. The function of waaF gene in Vibrio parahaemolyticus differs.