为进一步解析牛支原体（Mgcoplasma bovis）VspX蛋白的黏附特性，采用间接免疫荧光法检测VspX蛋白在M. bovis中的分布，通过黏附试验和抗体黏附抑制试验检测VspX蛋白的黏附性，采用ELISA方法进一步分析VspX蛋白和突变株结合纤连蛋白（Fn）的特性。结果显示，M. bovis VspX蛋白位于M. bovis菌体表面；重组VspX蛋白（rVspX）能黏附到EBL细胞表面，且M. bovis VspX基因缺失突变株（M. bovis ΔVspX）体外黏附EBL细胞能力与M. bovis野生株（M. bovis WT）相比显著下降，两个结果说明rVspX蛋白具有黏附特性；并且抗rVspX蛋白单抗能抑制M. bovis黏附EBL细胞，进而证实rVspX蛋白黏附的特异性；此外，rVspX蛋白与Fn呈剂量依赖性结合，且M. bovis ΔVspX结合Fn能力与M. bovis WT相比显著下降，证明M. bovis VspX蛋白与Fn为特异性结合，且Fn分布在EBL细胞表面。以上结果表明，M. bovis VspX蛋白是一种具有Fn结合特性的黏附相关蛋白，能通过EBL细胞外基质成分Fn介导其黏附EBL细胞。
Mycoplasma bovis is one of the serious pathogens causing bovine respiratory diseases. Meanwhile,M. bovis can cause a variety of clinical signs,including bronchopneumonia,mastitis,arthritis,genital tract inflammation,and tenosynovitis. Adhesion to airway epithelial cells is a key step for M. bovis colonization and invasion of cells. Adhesin is one of the main virulence factors of M. bovis. Research about attachment has become an important field of pathogenic mechanisms research of M. bovis. To further analyze the adhesion characteristics of the M. bovis VspX protein,experiments,including indirect immunofluorescence assay,adhesion assay of protein and mutant strain to embryonic bovine lung (EBL) cells,antibody inhibition adhesion detection,ELISA assay of protein and strains binding fibronectin (Fn),were carried out,to clarify the molecular mechanism of VspX protein adhesion. The results showed that the M. bovis VspX protein was located on the surface of the strain. The recombinant VspX protein (rVspX) was able to adhere to the embryonic bovine lung (EBL) cells. Compared with the M. bovis wild strain (M. bovis WT),the M. bovis VspX gene-deleted mutant strain (M. bovis ΔVspX) had a significantly decreased ability to adhere to EBL cells in vitro (P<0.05). The above results showed that the VspX protein was an adhesion-related protein. The anti-rVspX monoclonal antibody was able to inhibit the adhesion of M. bovis to EBL cells,which further confirmed the specificity of the adhesion. Also,the rVspX protein bound to Fn in a dose-dependent manner,and the M. bovis ΔVspX strain had a significantly lower adherence ability to Fn (P<0.05) than that of the M. bovis WT strain. These results further demonstrated the binding specificity of M. bovis VspX protein to Fn. And Fn was distributed on the surface of EBL cells. In summary,the study validated that M. bovis VspX protein was an adhesion-related protein with Fn binding properties,and the adhesion of VspX to EBL cells was mediated by the extracellular matrix component,Fn.