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

Abstract

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)

CLC Number:

S852.62

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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Development and Application of Monoclonal Antibodies against Mycoplasma mycoides subsp.Mycoides

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