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

Abstract

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

CLC Number:

S852.61

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.

References 31

[1]	LI Z Q, WANG S L, WEI S J, et al. Immunization with a combination of recombinant Brucella abortus proteins induces T helper immune response and confers protection against wild-type challenge in BALB/c mice[J]. Microb Biotechnol, 2022, 15(6):1811-1823.
[2]	SALVADOR-BESCÓS M, GIL-RAMÍREZ Y, ZÚÑIGA-RIPA A, et al. WadD, a new Brucella lipopolysaccharide core glycosyltransferase identified by genomic search and phenotypic characterization[J]. Front Microbiol, 2018, 9:2293.
[3]	MORENO E, BLASCO J M, LETESSON J J, et al. Pathogenicity and its implications in taxonomy:the Brucella and Ochrobactrum case[J]. Pathogens, 2022, 11(3):377.
[4]	KHURANA S K, SEHRAWAT A, TIWARI R, et al. Bovine brucellosis-a comprehensive review[J]. Vet Quart, 2021, 41(1):61-88.
[5]	YIN D H, BAI Q Q, WU X L, et al. Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein[J]. PLoS Negl Trop Dis, 2021, 15(8):e0009695.
[6]	DI BONAVENTURA G, ANGELETTI S, IANNI A, et al. Microbiological laboratory diagnosis of human brucellosis:an overview[J]. Pathogens, 2021, 10(12):1623.
[7]	ZHI F J, ZHOU D, LI J M, et al. Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro[J]. J Microbiol, 2020, 58(9):793-804.
[8]	REZAEI M, RABBANI-KHORASGANI M, ZARKESH-ESFAHANI S H, et al. Prediction of the omp16 epitopes for the development of an epitope-based vaccine against brucellosis[J]. Infect Disord Drug Targets, 2019, 19(1):36-45.
[9]	李杨. 布鲁菌OMP16对RAW264.7细胞凋亡与免疫活性的影响及其免疫原性研究[D]. 杨凌:西北农林科技大学, 2016.LI Y. The apoptosis and immunocompetence influence of Brucella OMP16 on RAW264. 7 and its immunogenicity study[D]. Yangling:Northwest A&F University, 2016. (in Chinese)
[10]	YANG X, HE Z P, ZHANG G X, et al. Evaluation of reactivity of monoclonal antibodies against Omp25 of Brucella spp.[J]. Front Cell Infect Microbiol, 2020, 10:145.
[11]	徐梦蔚, 宋武琦. 实验室水平单克隆抗体制备方法及相应纯化策略的研究进展[J]. 中国生物制品学杂志, 2022, 35(2):228-232, 238.XU M W, SONG W Q. Progress in research on methods for preparation of monoclonal antibody at laboratory level and relevant purification strategy[J]. Chinese Journal of Biologicals, 2022, 35(2):228-232, 238. (in Chinese)
[12]	蒋卉, 冯宇, 李筱英, 等. 布鲁氏菌4种高通量抗体检测方法的比较研究[J]. 畜牧兽医学报, 2021, 52(11):3208-3214.JIANG H, FENG Y, LI X Y, et al. Comparative research of four high throughput antibody detection methods for Brucella[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11):3208-3214. (in Chinese)
[13]	马帅, 李金积, 赵明, 等. 布鲁氏菌病研究进展[J]. 中国动物检疫, 2020, 37(7):73-79.MA S, LI J J, ZHAO M, et al. Research progress on brucellosis[J]. China Animal Health Inspection, 2020, 37(7):73-79. (in Chinese)
[14]	BAI Q Q, LI H, WU X L, et al. Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis[J]. Biochem Biophys Res Commun, 2021, 560:126-131.
[15]	IBAÑEZ A E, SMALDINI P, CORIA L M, et al. Unlipidated outer membrane protein Omp16(U-Omp16) from Brucella spp. as nasal adjuvant induces a Th1 immune response and modulates the Th2 allergic response to cow's milk proteins[J]. PLoS One, 2013, 8(7):e69438.
[16]	ALIZADEH H, DEZFULIAN M, RAHNEMA M, et al. Protection of BALB/c mice against pathogenic Brucella abortus and Brucella melitensis by vaccination with recombinant Omp16[J]. Iran J Basic Med Sci, 2019, 22(11):1302-1307.
[17]	田路路. 布鲁氏菌外膜蛋白16的原核表达及其抗体间接ELISA方法的建立[D]. 杨凌:西北农林科技大学, 2021.TIAN L L. Prokaryotic expression of Brucella OMP16 and establishment of indirect ELISA for its antibody[D]. Yangling:Northwest A&F University, 2021. (in Chinese)
[18]	林金香, 王心睿. 单克隆抗体制备技术的最新进展[J]. 中国免疫学杂志, 2021, 37(20):2544-2548.LIN J X, WANG X R. Recent advances on preparation technology of monoclonal antibody[J]. Chinese Journal of Immunology, 2021, 37(20):2544-2548. (in Chinese)
[19]	杜萌萌, 王小娟, 贺帅杰, 等. 犬CD19的表达及单克隆抗体制备[J]. 中国兽医科学, 2022, 52(4):458-465.DU M M, WANG X J, HE S J, et al. Expression and monoclonal antibodies preparation of canine CD19[J]. Chinese Veterinary Science, 2022, 52(4):458-465. (in Chinese)
[20]	熊流新, 黄健, 张涛, 等. 布鲁氏菌外膜蛋白Omp16的原核表达、纯化及多克隆抗体制备[J]. 现代医药卫生, 2018, 34(5):669-673.XIONG L X, HUANG J, ZHANG T, et al. Prokaryotic expression, purification and polyclonal antibody preparation of outer membrane proteins Omp16 from Brucella[J]. Journal of Modern Medicine and Health, 2018, 34(5):669-673. (in Chinese)
[21]	AKYA A, BOZORGOMID A, GHADIRI K, et al. Usefulness of blood parameters for preliminary diagnosis of brucellosis[J]. J Blood Med, 2020, 11:107-113.
[22]	PEREIRA C R, COTRIM DE ALMEIDA J V F, CARDOSO DE OLIVEIRA I R, et al. Occupational exposure to Brucella spp.:a systematic review and meta-analysis[J]. PLoS Negl Trop Dis, 2020, 14(5):e0008164.
[23]	GȽOWACKA P, ŻAKOWSKA D, NAYLOR K, et al. Brucella -virulence factors, pathogenesis and treatment[J]. Pol J Microbiol, 2018, 67(2):151-161.
[24]	董浩, 韩焘, 徐琦, 等. 3种国产布病抗体检测试剂盒的效果评价[J]. 中国兽医学报, 2020, 40(2):336-338.DONG H, HAN T, XU Q, et al. Evaluation of three domestic Brucellosis antibody test kits[J]. Chinese Journal of Veterinary Science, 2020, 40(2):336-338. (in Chinese)
[25]	高磊. 布鲁氏菌实时荧光定量PCR法的建立及对布鲁氏菌病诊断、疗效监测的研究[D]. 长春:吉林大学, 2020.GAO L. Establishment of real-time quantitative PCR for Brucella and research on diagnosis and efficacy monitoring of brucellosis[D]. Changchun:Jilin University, 2020. (in Chinese)
[26]	ÇIFTCI A,İÇA T, SAVAŞAN S, et al. Evaluation of PCR methods for detection of Brucella strains from culture and tissues[J]. Trop Anim Health Prod, 2017, 49(4):755-763.
[27]	AYDIN S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA[J]. Peptides, 2015, 72:4-15.
[28]	杨鑫. 应用布鲁菌外膜蛋白单克隆抗体检测布病患者外周血PBMCs中菌体抗原的研究[D]. 广州:南方医科大学, 2020.YANG X. Application of monoclonal antibodies to Brucella OMPs for antigen detection in PBMCs of brucellosis patients[D]. Guangzhou:Southern Medical University, 2020. (in Chinese)
[29]	姬智. 布鲁菌病几种血清学检测方法的比较与分析[D]. 呼和浩特:内蒙古农业大学, 2019.JI Z. Comparison and analysis of several serological detection methods for brucellosis[D]. Hohhot:Inner Mongolia Agricultural University, 2019. (in Chinese)
[30]	DUCROTOY M J, CONDE-ÁLVAREZ R, BLASCO J M, et al. A review of the basis of the immunological diagnosis of ruminant brucellosis[J]. Vet Immunol Immunopathol, 2016, 171:81-102.
[31]	田路路, 李会玲, 支飞杰, 等. 布鲁氏菌OMP16抗体的间接ELISA方法建立[J]. 动物医学进展, 2021, 42(3):30-35.TIAN L L, LI H L, ZHI F J, et al. Establishment of iELISA for detection of antibody against Brucella OMP16[J]. Progress in Veterinary Medicine, 2021, 42(3):30-35. (in Chinese)

Preparation and Preliminary Application of Monoclonal Antibody to Brucella Outer Membrane Protein 16

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