牛坏死杆菌的43 ku外膜蛋白在其黏附细胞中的作用初步分析

doi:10.11843/j.issn.0366-6964.2022.06.018

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (6): 1849-1857.doi: 10.11843/j.issn.0366-6964.2022.06.018

牛坏死杆菌的43 ku外膜蛋白在其黏附细胞中的作用初步分析

贺显晶, 蒋凯, 肖佳薇, 郭东华*

黑龙江八一农垦大学动物科技学院，大庆 163319

收稿日期:2021-05-10 出版日期:2022-06-23 发布日期:2022-06-25
通讯作者: 郭东华，主要从事奶牛腐蹄病发病机制及疫苗研究，E-mail：dh_guo@126.com
作者简介:贺显晶(1985-)，女，黑龙江佳木斯人，博士，高级实验师，主要从事动物厌氧菌病的综合防治研究，E-mail：xianjinghe@126.com
基金资助:
黑龙江省自然科学基金项目(LH2021C070);国家重点研发计划(2017YFD0502200);黑龙江八一农垦大学三纵科研支持计划青年创新人才项目(ZRCQC202103)

Preliminary Study on the Role of 43 ku Outer Membrane Protein of Fusobacterium necrophorum on Its Adhesion to Cells

HE Xianjing, JIANG Kai, XIAO Jiawei, GUO Donghua*

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2021-05-10 Online:2022-06-23 Published:2022-06-25

摘要/Abstract

摘要： 旨在明确牛坏死杆菌(Fusobacterium necrophorum)的43 ku外膜蛋白(43K OMP)在其黏附细胞中的作用，本研究将重组43K OMP蛋白和天然43K OMP蛋白与鼠乳腺上皮细胞共孵育，通过免疫荧光方法观察牛坏死杆菌43K OMP对鼠乳腺上皮细胞的黏附作用；同时利用天然蛋白竞争试验和抗体抑制试验明确43K OMP是否介导牛坏死杆菌与细胞的黏附，进一步利用牛坏死杆菌43K OMP基因缺失菌，评价43K OMP基因缺失对牛坏死杆菌黏附力的影响，阐明43K OMP在牛坏死杆菌黏附细胞中的作用。免疫荧光试验结果显示：天然43K OMP和重组43K OMP均能黏附于鼠乳腺上皮细胞表面，天然43K OMP与鼠乳腺上皮细胞或鼠肝细胞预孵育后，牛坏死杆菌黏附数量明显下降(P＜0.05)；牛坏死杆菌与43K OMP多抗或单抗预孵育后，黏附于鼠乳腺上皮细胞或鼠肝细胞的牛坏死杆菌数量显著降低(P＜0.05)；与牛坏死杆菌A25菌株相比，基因缺失菌A25Δ43K OMP黏附宿主细胞能力极显著下降(P＜0.01)，黏附率分别降低了94.4%和90.4%。因此，43K OMP在牛坏死杆菌黏附细胞中发挥关键性作用，深入研究其黏附机理将为揭示牛坏死杆菌致病机制提供理论基础。

关键词: 牛坏死杆菌, 43K OMP, 细菌黏附

Abstract: To clarify the role of the 43 ku outer membrane protein (43K OMP) in the adhesion of Fusobacterium necrophorum to cells, the mouse mammary epithelial cells and the recombinant protein or the natural protein were co-incubated in this study, and the adhesion of the 43K OMP of F.necrophorum was observed under a laser confocal microscope. Meanwhile, the adhesion inhibition assay with antibodies against the recombinant 43K OMP or the natural protein was used to determine the effect of the 43K OMP on the adhesion of F. necrophorum to host cells, and the effect of 43K OMP gene deletion on the adhesion of F. necrophorum to host cells was investigated using deletion mutant strain. Immunofluorescence results showed that the natural 43K OMP and recombinant 43K OMP could bind to the cell membrane of mouse mammary epithelial cells. When mouse mammary epithelial cells or mouse liver cells were preincubated with the natural 43K OMP, the adhesion of F. necrophorum significantly decreased (P < 0.05). When F. necrophorum was preincubated with polyclonal antibody or monoclonal antibody against the recombinant 43K OMP, the adhesion of F. necrophorum to mouse mammary epithelial cells or mouse liver cells significantly decreased compared with the negative serum control and the no antibody control (P < 0.05). The number of bacterial cells attached to cells was higher with F. necrophorum A25 strains than that of the mutant strain A25Δ43K OMP(P < 0.01), The 43K OMP gene deletion reduced the binding of F. necrophorum to mouse mammary epithelial cells or mouse liver cells by 94.4% and 90.4%. Therefore, the 43K OMP plays a key role in the adhesion of F. necrophorum to host cells, and further study of the adhesion mechanism will provide a theoretical basis for revealing the pathogenic mechanism of F. necrophorum.

Key words: Fusobacterium necrophorum, 43K OMP, bacterial adhesion

中图分类号:

S852.61

贺显晶, 蒋凯, 肖佳薇, 郭东华. 牛坏死杆菌的43 ku外膜蛋白在其黏附细胞中的作用初步分析[J]. 畜牧兽医学报, 2022, 53(6): 1849-1857.

HE Xianjing, JIANG Kai, XIAO Jiawei, GUO Donghua. Preliminary Study on the Role of 43 ku Outer Membrane Protein of Fusobacterium necrophorum on Its Adhesion to Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1849-1857.

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牛坏死杆菌的43 ku外膜蛋白在其黏附细胞中的作用初步分析

Preliminary Study on the Role of 43 ku Outer Membrane Protein of Fusobacterium necrophorum on Its Adhesion to Cells

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