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[Objective] To analyze the evolutionary conservation and structural characteristics of the heat shock protein GrpE from Mycoplasma bovis, elucidate its subcellular localization, and investigate its biological properties in mediating the adhesion process. [Methods] Primers were designed based on the GrpE gene sequence (GenBank accession number: CP002188.1) of Mycoplasma bovis PG45, and the prokaryotic expression vector pET-GrpE was constructed. Following gene sequencing, bioinformatics methods were employed to analyze the homology, phylogenetic relationships, physicochemical properties, and structural characteristics of GrpE. Following transformation of the recombinant plasmid and induced expression, the yielded recombinant GrpE protein was purified via nickel affinity chromatography, and then SDS-PAGE was conducted. The purified recombinant protein was used to immunize New Zealand White rabbits to generate polyclonal antibodies, with the antibody titer determined by ELISA and immunogenicity assessed via Western blotting. The subcellular localization of GrpE was examined via indirect indirect fluorescent antibody assay (IFA), ELISA, and Western blotting. The adhesion function of GrpE was validated through integrated IFA and ELISA. [Results] The prokaryotic expression vector pET-GrpE was successfully constructed in this study. Bioinformatics analysis revealed that the GrpE sequence was highly conserved in Mycoplasma bovis (with identity exceeding 95%). The encoded protein consisted of 341 amino acid residues, with no signal peptide and transmembrane domain but potential N-glycosylation and phosphorylation sites. SDS-PAGE results confirmed the successful expression of GrpE in a soluble form. Polyclonal antibodies generated via the purified recombinant protein exhibited a titer of 1:16 000. Western blotting analysis further verified the strong immunogenicity of the GrpE protein. Localization studies using IFA, ELISA, and Western blotting indicated that GrpE is distributed in both the cell membrane and the cytoplasm, with predominant distribution observed on the membrane surface. Importantly, the anti-GrpE polyclonal antiserum significantly inhibited the adhesion of Mycoplasma bovis to embryonic bovine lung (EBL) cells. Furthermore, binding assays demonstrated that the interaction between GrpE and host cell membrane proteins is dose-dependent, and this binding was inhibited by the polyclonal antibody (P<0.001). [Conclusion] GrpE is identified as a highly conserved novel adhesion of Mycoplasma bovis that directly participates in the adhesion to host cells, providing a key molecular target for elucidating the pathogenic mechanism of Mycoplasma bovis.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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