布鲁氏菌外膜蛋白16单克隆抗体的制备及初步应用

doi:10.11843/j.issn.0366-6964.2023.05.029

摘要/Abstract

摘要： 旨在制备布鲁氏菌外膜蛋白16(OMP16)单克隆抗体,建立OMP16抗体竞争ELISA方法,为布病临床防控提供免疫血清学检测手段。本研究选取保守性高、免疫原性良好的布鲁氏菌OMP16,经原核表达获得携带GST或His标签的重组OMP16(rOMP16)。利用杂交瘤技术筛选可稳定分泌OMP16特异性单克隆抗体的杂交瘤细胞株,经亚型鉴定、细胞核型分析和抗体交叉反应性试验分析单抗性质。制备小鼠腹水并纯化,方阵滴定法建立布鲁氏菌OMP16抗体竞争ELISA方法。结果表明,成功构建OMP16原核表达系统,经表达、纯化获得rGST-uOMP16和rHis-OMP16。筛选到1株遗传稳定的OMP16特异性杂交瘤细胞株,命名为B7;经鉴定,该抗体亚型为IgM-κ型,可识别目的蛋白,与标签蛋白无交叉反应。建立了布鲁氏菌OMP16抗体竞争ELISA方法,该方法与试管凝集试验检测结果相比总体符合率为90.53%,显示出良好的一致性。本研究有望为布鲁氏菌病的临床防控提供特异性高、敏感性好的免疫血清学检测方法。

关键词: 布鲁氏菌, 外膜蛋白16, 原核表达, 单克隆抗体, 竞争ELISA

Abstract: The aim of this study was to prepare monoclonal antibodies against Brucella outer membrane protein 16 (OMP16), establish a competitive ELISA method for OMP16 antibody, and provide an immune serological detection method for clinical prevention and control of brucellosis. In this study, Brucella OMP16 with high conservation and good immunogenicity was selected, and recombinant OMP16 carrying GST or His tag was obtained by prokaryotic expression. Hybridoma cell lines stably secreting OMP16 specific monoclonal antibodies were screened by hybridoma technique. The properties of monoclonal antibodies were analyzed by subtype identification, hybridoma cell karyotype analysis and antibody cross-reactivity test. Preparation and purification of mouse ascites and establishment of competitive ELISA for Brucella OMP16 antibody by square titration. The prokaryotic expression system of OMP16 was constructed, and rGST-uOMP16 and rHis-OMP16 were obtained by expression and purification. A genetically stable OMP16-specific hybridoma cell line was screened and named B7. After identification, the antibody subtype was identified as IgM-κ, recognizes the target protein, and has no cross reaction with the tag protein. The Brucella OMP16 antibody competition ELISA method was established. Compared with the results of the standard tube agglutination test, the overall coincidence rate of this method was 90.53%, showing good consistency. It is expected to provide an immune serological detection method with high specificity and good sensitivity for the clinical prevention and control of brucellosis.

Key words: Brucella, outer membrane protein 16, prokaryotic expression, monoclonal antibody, competitive ELISA

中图分类号:

S852.61

翟云逸, 袁野, 李俊玫, 田路路, 刁梓洋, 李彬, 陈家露, 周栋, 靳亚平, 王爱华. 布鲁氏菌外膜蛋白16单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2083-2091.

ZHAI Yunyi, YUAN Ye, LI Junmei, TIAN Lulu, DIAO Ziyang, LI Bin, CHEN Jialu, ZHOU Dong, JIN Yaping, WANG Aihua. Preparation and Preliminary Application of Monoclonal Antibody to Brucella Outer Membrane Protein 16[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2083-2091.

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