牛源坏死梭杆菌43K外膜蛋白的黏附特性研究

doi:10.11843/j.issn.0366-6964.2023.02.029

摘要/Abstract

摘要： 旨在明确牛源坏死梭杆菌43K OMP的黏附特性。将43K OMP基因克隆连接至pET-32a载体，转化入大肠杆菌BL21 DE3中，通过IPTG诱导进行原核表达，应用黏附试验、天然蛋白竞争试验、抗体抑制试验和蛋白酶水解试验，明确牛源坏死梭杆菌43K OMP的黏附性，同时将纯化的重组蛋白和提取的天然43K OMP与牛子宫内膜细胞和牛乳腺上皮细胞共孵育，观察43K OMP对细胞的黏附作用。结果显示：43K OMP基因克隆到pET-32a载体中，随后在大肠杆菌BL21 DE3以包涵体形式成功表达；携带重组质粒（H2019）的大肠杆菌经IPTG诱导后，与空载体对照相比，与宿主细胞的结合显著增强（P<0.05）；天然43K OMP与细胞共孵育后，黏附细胞的细菌数量显著降低（P<0.05）；H2019与43K OMP多抗或单抗预孵育后，显著降低黏附宿主细胞的细菌数量（P<0.05）；经不同浓度蛋白酶K处理H2019后，黏附细胞的细菌数量显著降低（P<0.05），且与蛋白酶K浓度呈现剂量依赖关系。同时，43K OMP天然蛋白和重组蛋白能黏附于牛子宫内膜细胞和乳腺上皮细胞表面。牛源坏死梭杆菌43K OMP能黏附宿主细胞，43K OMP作为一种黏附蛋白有助于牛源坏死梭杆菌对宿主细胞的黏附作用。

关键词: 牛源坏死梭杆菌, 43K OMP, 细菌黏附, 上皮细胞

Abstract: Herein, we attempted to clarify the adhesion characterization of 43K OMP of bovine Fusobacterium necrophorum.In this study, 43K OMP gene of F. necrophorum was cloned into pET-32a vector and the proteins were subsequently expressed on the surface of E. coli BL21 DE3 cells. The attachment assay, antibody inhibition attachment assay, native protein inhibition assay and proteinase treatment assay were used to determine the adhesion characterization of 43K OMP in bovine F. necrophorum. Meanwhile, the recombinant protein and naive 43K OMP were co-incubated with bovine endometrial epithelial cells or bovine mammary epithelial cells(MAC-T), and then evaluated the adhesion effect of 43K OMP of F. necrophorum to bovine epithelial cells. The 43K OMP gene was cloned into pET-32a vector and the recombinant 43K OMP in the form of an inclusion body was present in the bacterial precipitation. When E. coli carrying the recombinant plasmid (H2019) was induced by IPTG, there was significant enhancement in the binding to host cells compared to the E. coli carrying control vector only(P<0.05). When fixed cells were incubated with purified native 43K OMP, H2019 showed lower levels of binding(P<0.05). Pre-incubation of induced H2019 with polyclonal antibodies or monclonal antibodies against the 43K OMP reduced the binding amount to host cells(P<0.05). The adhesion of F. necrophorum was significantly decreased after proteinase K treatment(P<0.05), and dose-dependent relationship was shown between the decrease degree of adhesionto host cells and the concentrations of proteinase K. At the same time, the native 43K OMP and recombinant 43K OMP could bind to bovine endometrial epithelial cells or MAC-T cells. The 43K OMP of bovine F. necrophorum possessed the ability of adhesion to bovine host cells, and the 43K OMP acted as an adhesion protein to improve the binding ability of bovine F. necrophorum to host cells.

Key words: bovine Fusobacterium necrophorum, 43K OMP, bacterial adhesion, epithelial cells

中图分类号:

S852.61

贺显晶, 刘娇, 王志慧, 武瑞, 郭东华. 牛源坏死梭杆菌43K外膜蛋白的黏附特性研究[J]. 畜牧兽医学报, 2023, 54(2): 726-735.

HE Xianjing, LIU Jiao, WANG Zhihui, WU Rui, GUO Donghua. Adhesion Characterization of 43K OMP in Fusobacterium necrophorum subsp. necrophorum[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 726-735.

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