抗丝状支原体丝状亚种单克隆抗体的制备及初步应用

doi:10.11843/j.issn.0366-6964.2022.07.025

摘要/Abstract

摘要： 旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb)，为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体。本研究利用生物信息学技术分析了Mmm国内分离株Ben-1不同传代株的全基因组序列，选取M0071蛋白作为研究对象。将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠，通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株。进一步制备单抗腹水并纯化，利用Western blot方法对该单抗进行特异性鉴定，同时测定其抗体效价和抗体亚类。随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力。结果表明成功获得1株单克隆细胞株3C4A1，将其分泌抗体命名为MAb 3C4A1。特异性结果表明，MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应，而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应。抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链。经间接ELISA测定其抗体效价为1∶256 000。IFA试验结果表明，MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应，而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应，特异性良好。本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性，可作为CBPP病原免疫学诊断的工具，为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料。

关键词: 丝状支原体丝状亚种, M0071蛋白, 单克隆抗体, 间接免疫荧光检测

Abstract: The aim of this study was to develop monoclonal antibodies (MAbs) against Mycoplasma mycoides subsp. Mycoides (Mmm), and to provide a specific monoclonal antibody (MAb) for immunological methods for detection of Mmm, the causative agent of contagious bovine pleuropneumonia (CBPP). In this study, the protein M0071 was selected based on bioinformatics analysis of the whole genomes of five representative generations of Mmm Ben-1 isolates. A soluble fusion protein M0071 was expressed in prokaryotic expression system, the purified recombinant protein M0071 (rM0071) was used as an immunogen to immunize BALB/c mice. The hybridoma 3C4A1 stably secreting MAbs against rM0071 protein, was produced after screening by limited dilution method and an indirect ELISA method. MAb 3C4A1 were purified from mouse ascites. The specificity of the MAbs was identified using Western blot method, the titer and isotype of the purified MAbs were also determined. Further, indirect immunofluorescence (IFA) assay was used to evaluate the potency of the MAbs as primary antibodies for the detection of Mmm infection. One hybridoma 3C4A1 was successfully obtained, and its secreted antibody was named MAb 3C4A1. Western blot results showed that MAb 3C4A1 specifically reacted with Mmm isolates and type strain, while no reaction occurred with Mycoplasma capricolumsubsp.capripneumoniae, Mycoplasma mycoides subsp.capri, Mycoplasma bovirhinis, Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma leachii and bovine Pasteurella type A. Isotype analysis showed that MAb 3C4A1 belonged to IgG1, and the light chains belonged to κ. The ascites titer was 1:256 000. IFA results showed that MAb 3C4A1 only reacted with EBL cells infected with Mmm, while did not react with Mycoplasma bovirhinis, Mycoplasma agalactiae and Mycoplasma bovis, showing excellent specificity. MAb 3C4A1 showed excellent specificity and immunoreactivity, and could be used as a valuable tool for immunological diagnosis of CBPP pathogens, which provides basic material for further development of differential diagnostic techniques of CBPP pathogens.

Key words: Mycoplasma mycoidessubsp.Mycoides, M0071 protein, monoclonal antibody, indirect immunofluorescence assay (IFA)

中图分类号:

S852.62

曾金红, 徐青元, 沙玮萍, 吴爽, 李媛, 辛九庆, 王秀梅. 抗丝状支原体丝状亚种单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2022, 53(7): 2290-2299.

ZENG Jinhong, XU Qingyuan, SHA Weiping, WU Shuang, LI Yuan, XIN Jiuqing, WANG Xiumei. Development and Application of Monoclonal Antibodies against Mycoplasma mycoides subsp.Mycoides[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2290-2299.

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