牛支原体贵州株黏附相关蛋白的初步鉴定

doi:10.11843/j.issn.0366-6964.2024.04.029

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (4): 1672-1683.doi: 10.11843/j.issn.0366-6964.2024.04.029

牛支原体贵州株黏附相关蛋白的初步鉴定

罗小芬¹, 谢晓东¹, 赵超¹, 胡茜¹, 王永璇¹, 冉芳菲¹, 胡鹏飞³, 文明^1,2, 朱二鹏^1,2*, 程振涛^1,2*

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州省动物疫病与兽医公共卫生重点实验室, 贵阳 550025;
3. 盘州市动物疫病预防控制中心, 盘州 553500

收稿日期:2023-07-18 出版日期:2024-04-23 发布日期:2024-04-26
通讯作者: 朱二鹏,主要从事动物疫病研究,E-mail:zhu13782701756@126.com;程振涛,主要从事动物疫病研究,E-mail:chengzhentao@sohu.com
作者简介:罗小芬(1999-)，女，苗族，贵州长顺人，硕士生，主要从事动物疫病研究，E-mail:1944079106@qq.com
基金资助:
贵州省科技计划项目(黔科合支撑[2021]一般161号；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项目(贵大[省]创字2022[099]号)

Initial Identification of Adhesion-related Proteins of Mycoplasma bovis of Guizhou Strains

LUO Xiaofen¹, XIE Xiaodong¹, ZHAO Chao¹, HU Qian¹, WANG Yongxuan¹, RAN Fangfei¹, HU Pengfei³, WEN Ming^1,2, ZHU Erpeng^1,2*, CHENG Zhentao^1,2*

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Guizhou Provincial Key Laboratory of Animal Diseases and Veterinary Public Health, Guiyang 550025, China;
3. Panzhou Animal Disease Prevention and Control Center, Panzhou 553500, China

Received:2023-07-18 Online:2024-04-23 Published:2024-04-26

摘要/Abstract

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。

关键词: 牛支原体, 黏附蛋白, 原核表达, 筛选

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 investigate 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, subcellular 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 confirmed 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 inhibits the adhesion of M. bovis to EBL cells. This finding provides a basis of reference for the selection of target proteins for the diagnosis of M. bovis-associated diseases and the development of preventive vaccines.

Key words: Mycoplasma bovis, adhesion protein, prokaryotic expression, screening

中图分类号:

S852.62

罗小芬, 谢晓东, 赵超, 胡茜, 王永璇, 冉芳菲, 胡鹏飞, 文明, 朱二鹏, 程振涛. 牛支原体贵州株黏附相关蛋白的初步鉴定[J]. 畜牧兽医学报, 2024, 55(4): 1672-1683.

LUO Xiaofen, XIE Xiaodong, ZHAO Chao, HU Qian, WANG Yongxuan, RAN Fangfei, HU Pengfei, WEN Ming, ZHU Erpeng, CHENG Zhentao. Initial Identification of Adhesion-related Proteins of Mycoplasma bovis of Guizhou Strains[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1672-1683.

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牛支原体贵州株黏附相关蛋白的初步鉴定

Initial Identification of Adhesion-related Proteins of Mycoplasma bovis of Guizhou Strains

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