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【目的】阐明霍乱弧菌DsbA蛋白(VcDsbA)在生物被膜形成过程中的作用。【方法】采用Overlapping PCR的方法构建VcdsbA基因敲除质粒p MH524;采用同源重组和基因克隆的方法对霍乱弧菌dsbA(vc0034)基因进行敲除和回补;通过结晶紫染色实验,比较野生株(WT)、dsbA突变株(ΔdsbA)和回补菌株(CΔdsbA)的生物被膜形成差异;用荧光素酶基因作为报告基因分析与生物被膜形成相关的甘露糖敏感血凝素纤毛合成蛋白(Mannose-sensitive hemagglutinin,pili biogenesis protein,MSHA)在霍乱弧菌WT、ΔdsbA和CΔdsbA中表达水平的区别。【结果】成功构建霍乱弧菌dsbA基因缺失突变株和回补株;与WT相比,ΔdsbA生物被膜形成能力显著下降,且msh操纵子的表达水平显著降低。【结论】VcDsbA可能通过影响其它调控因子直接或间接增强霍乱弧菌MSHA的生物合成,从而促进霍乱弧菌生物被膜的形成。本文为进一步研究DsbA在霍乱弧菌生物被膜形成中的调控机制奠定了基础。
【Objective】 To clarify the role of VcDsbA in the biofilm formation process. 【Method】 VcdsbA gene knockout plasmid p MH524 was constructed by Overlapping PCR. Knockout and replenishment of Vibrio cholera dsbA (vc0034) gene were carried out by homologous recombination and gene cloning. The crystal violet staining assay was used to compare the wild type Differences were observed in the biofilms of WT, dsbA mutant (ΔdsbA) and reference strain (CΔdsbA); mannose-sensitive hemagglutinin cili synthesis protein associated with biofilm formation was analyzed using the luciferase gene as a reporter -sensitive hemagglutinin, pili biogenesis protein, MSHA) in V. cholerae WT, ΔdsbA and CΔdsbA. 【Result】 The dsbA gene deletion mutants and the complemented strains of V. cholerae were successfully constructed. Compared with WT, the ability of ΔdsbA biofilm formation decreased significantly and the expression level of msh operon was significantly reduced. 【Conclusion】 VcDsbA may directly or indirectly enhance the biosynthesis of Vibrio cholerae MSHA by affecting other regulators, thereby promoting the formation of Vibrio cholerae biofilm. This article lays the foundation for further research on the regulatory mechanism of DsbA in the biofilm formation of V. cholerae.