摘要
牛支原体(Mycoplasma bovis,M.bovis)是引起牛乳腺炎和小牛肺炎等多种牛病的重要病原体.黏附蛋白通常被认为在该生物体的致病机制中起着核心作用.因此,本研究旨在以牛支原体株贵州株(GZ-2)的4个假定蛋白为研究对象,分别命名为M27、M32、M498、M663;分别在大肠杆菌感受态细胞DE3(BL21)中进行原核表达及纯化、亚细胞定位及黏附性研究.结果显示:成功表达和纯化重组M27、M32、M498和M663蛋白;M27、M32和M498蛋白均在牛支原体总蛋白、胞膜蛋白与胞质蛋白中均出现特异印迹条带,而M663未被检测到;共聚焦激光扫描显微镜下的免疫染色结果显示,M27、M32、M498和M663蛋白和牛支原体都能黏附在胚胎牛肺细胞(EBL)上,兔抗M27、M32、M498和M663血清抗体能够抑制蛋白M27、M32、M498和M663对EBL细胞的黏附,也能抑制牛支原体对EBL细胞的黏附.流式细胞术分析结果与其一致,得到了进一步证实.综上所述,M27、M32和M498蛋白是牛支原体的黏附相关蛋白,而M663在Western blot中未被检测到,但它仍能黏附在EBL细胞上,并且其抗血清可抑制牛支原体对EBL细胞的黏附.这为诊断牛支原体相关疾病和开发预防性疫苗选择目标蛋白提供参考依据.
Abstract
Mycoplasma bovis(M.bovis)is an important pathogen that causes a variety of bovine diseases including bovine mastitis and calf pneumonia.Adhesion proteins are often thought to play key roles in the pathogenic mechanisms of M.bovis.Therefore,this study aimed to investi-gate four putative proteins of M.bovis Guizhou strain(GZ-2),namely M27,M32,M498,and M663,through prokaryotic expression in Escherichia coli BL21(DE3)and purification,subcel-lular localization and adhesion studies.The results showed that recombinant M27,M32,M498 and M663 proteins were successfully expressed and purified;M27,M32 and M498 proteins all showed specific blotting bands in total proteins,cytosolic and cytoplasmic proteins of M.bovis,whereas M663 was undetectable;Immunostaining results showed that M27,M32,M498 and M663 proteins as well as M.bovis adhered to embryonic bovine lung(EBL)cells under confocal laser scanning microscopy,and the rabbit anti-M27,M32,M498 and M663 antibodies were able to inhibit the adherence of these proteins and M.bovis to the EBL cells.This was further con-firmed by flow cytometry analysis with identical results.In conclusion,M27,M32 and M498 proteins are adhesion-associated proteins of M.bovis;of note,although M663 proteins were not detected in the Western blot analysis,it still is able to adhere to EBL cells and simultaneously in-hibits the adhesion of M.bovis to EBL cells.This finding provides a basis of reference for the se-lection of target proteins for the diagnosis of M.bovis-associated diseases and the development of preventive vaccines.
基金项目
贵州省科技计划(黔科合支撑[2021]一般161号)
贵州省科技计划(黔科合支撑[2023]一般077号)
贵州省优秀青年科技人才项目(黔科合平台人才[2021]5646号)
大学生创新创业训练计划(贵大[省]创字2022[099]号)
维普
万方数据