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 establish a Western blotting method for Vibrio parahaemolyticus (VP). Methods The VPA1405 gene was cloned into pET-28a (+) vector and transformed into E. coli BL21 for insoluble expression. The VPA1405 protein was purified under denaturing conditions. The prepared inclusion body solution was directly immunized with rabbit to obtain polyclonal antibody, The difference in expression level of VPA1405 protein in wild type (WT) and opaR knockout (ΔopaR) was detected by Western blotting. Results The protein of 6 biofilm-related genes was successfully expressed and purified. Western blotting showed that OpaR regulators positively regulated the expression of VPA1405, which was consistent with the phenotypic results. Conclusion The VP Western blot experiment platform was successfully established, which can be used for the subsequent regulation of VP biofilm-related genes.