作　　者： ; ; ; ; ; ; ; ;

机构地区： 重庆医科大学公共卫生与管理学院

出　　处： 《军事医学》 2013年第2期118-121,125,共5页

摘　　要： 目的建立副溶血弧菌(Vibrio parahaemolyticus,VP)的Western印迹方法。方法将VPA1405基因克隆到pET-28a(+)载体,转入大肠杆菌BL21中进行不可溶性表达,在变性条件下纯化得到VPA1405蛋白,制备的包涵体溶液直接免疫兔得到多克隆抗体,进而利用Western印迹检测VPA1405蛋白在野生株(WT)和opaR基因敲除株(ΔopaR)中表达水平的差异。结果成功表达纯化了6个与生物膜相关基因的蛋白;Western印迹结果显示OpaR调控子对VPA1405基因的表达呈正调控,这与表型实验结果相符。结论成功建立了VP的Western印迹实验平台,该平台可用于后续VP生物膜相关基因的调控研究。 Objective To construct a Western blot method for Vibrio parahaemolyticus. Methods The entire coding region of VPA1405 was cloned into pET-28a （ ＋ ） vector, and then the recombinant plasmid pET28a（ ＋ ）-VPA1405 was transformed into Escherichia coli BL21 to express the insoluble His-VPA1405 protein. Polyclonal antibodies were obtained by immunizing rabbits with His-VPA1405, then the VPA1405 expression levels in the wild type strain （WT） and the opaR mutant strain （AopaR） were analyzed using the Western blot method. Results Six biofilm formation related proteins were successfully expressed. The expression of VPA1405 was positively regulated by OpaR, which was consistent with the results of phenotype experiments. Conclusion The Western blotting method is successfully constructed, which can be used for the study of biofilm formation mechanisms in V. parahaemolytlcus.

关 键 词： 副溶血弧菌 生物膜 印迹

领　　域： [生物学]