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      2. 牛支原體VspX蛋白黏附特性解析
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        國家重點研發計劃項目(2018YFD0501802);國家現代農業(肉牛/牦牛)產業技術體系項目(CARS-37);全國農業科研杰出人才及其“牛病防治基礎與技術創新團隊”專項;河北省自然科學青年基金項目(C2018402137)


        Adhesion characteristics of Mycoplasma bovis VspX protein
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          摘要:

          為進一步解析牛支原體(Mgcoplasma bovis)VspX蛋白的黏附特性,采用間接免疫熒光法檢測VspX蛋白在M. bovis中的分布,通過黏附試驗和抗體黏附抑制試驗檢測VspX蛋白的黏附性,采用ELISA方法進一步分析VspX蛋白和突變株結合纖連蛋白(Fn)的特性。結果顯示,M. bovis VspX蛋白位于M. bovis菌體表面;重組VspX蛋白(rVspX)能黏附到EBL細胞表面,且M. bovis VspX基因缺失突變株(M. bovis ΔVspX)體外黏附EBL細胞能力與M. bovis野生株(M. bovis WT)相比顯著下降,兩個結果說明rVspX蛋白具有黏附特性;并且抗rVspX蛋白單抗能抑制M. bovis黏附EBL細胞,進而證實rVspX蛋白黏附的特異性;此外,rVspX蛋白與Fn呈劑量依賴性結合,且M. bovis ΔVspX結合Fn能力與M. bovis WT相比顯著下降,證明M. bovis VspX蛋白與Fn為特異性結合,且Fn分布在EBL細胞表面。以上結果表明,M. bovis VspX蛋白是一種具有Fn結合特性的黏附相關蛋白,能通過EBL細胞外基質成分Fn介導其黏附EBL細胞。

          Abstract:

          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.

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        陳曦,劉東明,張慧,朱習芳,陳穎鈺,胡長敏,郭愛珍.牛支原體VspX蛋白黏附特性解析[J].華中農業大學學報,2020,39(4):77-84

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        • 收稿日期:2020-04-26
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        • 在線發布日期: 2020-07-30
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