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目的:IpaH家族是福氏志贺菌Ⅲ型分泌系统(T3SS)效应子的成员,但功能不清,特别是对IpaH4.5的研究未见报道。本研究拟探讨IpaH4.5的功能。方法:克隆了福氏志贺菌2a 301株的IpaH4.5基因,构建了带Flag和GFP标签的IpaH4.5真核表达载体。通过Western印迹和共聚焦确定IpaH4.5在真核细胞293T中的表达和定位,通过报道基因的方法观察IpaH4.5对宿主细胞NF-κB途径的影响。结果:Western印迹证实IpaH4.5在真核细胞中有表达,激光共聚焦确定IpaH4.5在真核细胞中定位于细胞膜和细胞核,通过报道基因观察到它可以抑制宿主细胞的NF-κB途径。结论:发现IpaH4.5可以抑制NF-κB途径,为进一步研究IpaH4.5的功能奠定了基础。
OBJECTIVE: The IpaH family is a member of the Shigella flexneri type III secretion system (T3SS) effector, but its function is poor, especially for IpaH4.5. This study intends to explore the function of IpaH4.5. Methods: The IpaH4.5 gene of Shigella flexneri 2a 301 was cloned and the IpaH4.5 eukaryotic expression vector with Flag and GFP tag was constructed. The expression and localization of IpaH4.5 in eukaryotic 293T cells were determined by Western blotting and confocal analysis. The effect of IpaH4.5 on NF-κB pathway in host cells was observed by reporter gene method. Results: Western blotting confirmed that IpaH4.5 was expressed in eukaryotic cells. Confocal laser scanning confocal microscope showed that IpaH4.5 localized in the cell membrane and nucleus in eukaryotic cells. The reporter gene was found to inhibit the NF-κB pathway in host cells. CONCLUSION: It was found that IpaH4.5 can inhibit the NF-κB pathway, which laid the foundation for further study on the function of IpaH4.5.