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【目的】副溶血弧菌通过分泌多种毒力因子引起急性胃肠炎,其中Ⅵ型分泌系统(T6SS1和T6SS2)是近年来新发现的一种毒力因子分泌系统,但该系统如何受到调控的、如何影响细胞粘附等问题在副溶血弧菌中尚不清楚。本研究的目的是为了阐明副溶血弧菌调控因子VPA1045和VPA1049对T6SS2分泌系统在转录、翻译和翻译后水平的调控作用。【方法】利用同源重组方法构建Vpa1045和Vpa1049缺失株,荧光定量方法检测T6SS2转位因子hcp2的转录水平差异,免疫印迹分析细菌菌体中Hcp2的表达与上清中Hcp2的跨膜转位差异。【结果】副溶血弧菌VPA1045和VPA1049的基因结构中均含有Che-Y结构域,属于二元调控因子的反应调节因子。缺失Vpa1045和Vpa1049后,菌体中的Hcp2表达量和基因组中hcp2的转录水平差异不显著,细菌上清中的Hcp2转位量和对HeLa细胞的细胞粘附率与亲本株HZ相比显著下降。【结论】二元调控因子VPA1045和VPA1049通过上调Hcp2的转位量从而翻译后上调副溶血弧菌T6SS2。
【Objective】 Vibrio parahaemolyticus causes acute gastroenteritis through secretion of various virulence factors, of which type Ⅵ secretion system (T6SS1 and T6SS2) is a newly discovered virulence factor secretion system in recent years, but how the system is regulated How to affect the cell adhesion and other issues in Vibrio parahaemolyticus is not clear. The aim of this study was to elucidate the regulatory effect of Vibrio parahaemolyticus VPA1045 and VPA1049 on the transcription, translation and posttranslational levels of the T6SS2 secretion system. 【Method】 The homologous recombination method was used to construct the Vpa1045 and Vpa1049 deletion strains. Fluorescence quantitative method was used to detect the difference of transcription level of T6SS2 translocation factor hcp2. Western blotting was used to analyze the difference of Hcp2 expression in bacterial cells and transmembrane translocation of Hcp2 in supernatant . 【Result】 The results showed that both the Vibrio parahaemolyticus VPA1045 and VPA1049 contained Che-Y domain, which is a response regulator of binary regulatory factors. After deletion of Vpa1045 and Vpa1049, the expression level of Hcp2 in the cells was not significantly different from that of the hcp2 in the genome. The Hcp2 translocation and the cell adhesion rate to HeLa cells in the bacterial supernatant were significantly decreased compared with the parent strain HZ . 【Conclusion】 The binary regulatory factors VPA1045 and VPA1049 up-regulated the translocation of Hcp2 to post-translationally up-regulate Vibrio parahaemolyticus T6SS2.