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.

References 31

[1]	ABDO E M, NICOLET J, MISEREZ R, et al. Humoral and bronchial immune responses in cattle experimentally infected with Mycoplasma mycoides subsp. mycoides small colony type[J]. Vet Microbiol, 1998, 59(2-3):109-122.
[2]	吴移谋,叶元康.支原体学[M]. 2版.北京:人民卫生出版社, 2008:237-242.WU Y M, YE Y K. Mycoplasmology[M]. 2nd ed. Beijing:People's Medical Publishing House, 2008:237-242.(in Chinese)
[3]	DI TEODORO G, MARRUCHELLA G, DI PROVVIDO A, et al. Contagious bovine pleuropneumonia:A comprehensive overview[J]. Vet Pathol, 2020, 57(4):476-489.
[4]	DUDEK K, SZACAWA E, NICHOLAS R A J. Recent developments in vaccines for bovine mycoplasmoses caused by Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides[J]. Vaccines, 2021, 9(6):549.
[5]	REGALLA J, CAPORALE V, GIOVANNINI A, et al. Manifestation and epidemiology of contagious bovine pleuropneumonia in Europe[J]. Rev Sci Tech, 1996, 15(4):1309-1329.
[6]	ARFI Y, MINDER L, DI PRIMO C, et al. MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G[J]. Proc Natl Acad Sci U S A, 2016, 113(19):5406-5411.
[7]	叶德成,吴海杰.牛传染性胸膜肺炎研究进展[J].中国畜牧兽医文摘, 2018, 34(6):194.YE D C, WU H J. Research progress of contagious bovine pleuropneumonia[J]. China Veterinary Animal Husbandry Digest, 2018, 34(6):194.(in Chinese)
[8]	沈青春,韩明远,王芳,等.牛传染性胸膜肺炎研究进展[J].中国奶牛, 2010(8):48-51.SHEN Q C, HAN M Y, WANG F, et al. Progress on containions bovine pleuropneumonia[J]. China Dairy Cattle, 2010(8):48-51.(in Chinese)
[9]	WINDSOR R S. The eradication of contagious bovine pleuropneumonia from south western Africa:a plan for action[J]. Ann N Y Acad Sci, 2000, 916:326-332.
[10]	TARDY F, GAURIVAUD P, MANSO-SILVÁN L, et al. Extended surveillance for CBPP in a free country:challenges and solutions regarding the potential Caprine reservoir[J]. Prev Vet Med, 2011, 101(1-2):89-95.
[11]	BÖLSKE G, MSAMI H M, GUNNARSSON A, et al. Contagious bovine pleuropneumonia in northern Tanzania, culture confirmation and serological studies[J]. Trop Anim Health Prod, 1995, 27(4):193-201.
[12]	ABDELA N, YUNE N. Seroprevalence and distribution of contagious bovine pleuropneumonia in Ethiopia:Update and critical analysis of 20 years (1996-2016) reports[J]. Front Vet Sci, 2017, 4:100.
[13]	VARELA F, INÁCIO J, BOTELHO A. Molecular diversity assessed by VNTR and IS1296 typing of historical Mycoplasma mycoides subsp. mycoides SC strains[J]. Vet Microbiol, 2010, 146(3-4):295-302.
[14]	LI Y, WANG Y, WANG R, et al. Changes in pathogenicity and immunogenicity of Mycoplasma mycoides subsp. mycoides strains revealed by comparative genomics analysis[J]. Sci Rep, 2016, 6:19081.
[15]	XIN J Q, LI Y, NICHOLAS R A J, et al. A history of the prevalence and control of contagious bovine pleuropneumonia in China[J]. Vet J, 2012, 191(2):166-170.
[16]	陈金水,翟旭久.牛传染性胸膜肺炎的血清学诊断[J].中国兽医科技, 1990(9):38-39.CHEN J S, ZHAI X J. Serological diagnosis of contagious bovine pleuropneumonia[J]. Chinese Journal of Veterinary Science and Technology, 1990(9):38-39.(in Chinese)
[17]	MUSA J A, BALE J O O, KAZEEM H M, et al. Molecular detection of Nigerian field isolates of Mycoplasma mycoides subsp. mycoides as causative agents of contagious bovine pleuropneumonia[J]. Int J Vet Sci Med, 2016, 4(2):46-53.
[18]	MAIR G, VILEI E M, WADE A, et al. Isothermal loop-mediated amplification (LAMP) for diagnosis of contagious bovine pleuropneumonia[J]. BMC Vet Res, 2013, 9:108.
[19]	PILO P, FREY J, VILEI E M. Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC[J]. Vet J, 2007, 174(3):513-521.
[20]	世界动物卫生组织(OIE). OIE陆生动物诊断试验与疫苗手册[M].农业部兽医局,译. 7版.北京:中国农业出版社, 2012.The World Organization for Animal Health (OIE). OIE handbook of diagnostic tests and vaccines for terrestrial animals[M]. Veterinary Bureau of the Ministry of Agriculture, trans. 7th ed. Beijing:China Agriculture Press, 2012.(in Chinese)
[21]	纪燕,熊祺琰,韦艳娜,等.猪鼻支原体黏附宿主细胞间接免疫荧光检测方法的建立[J].中国人兽共患病学报, 2014, 30(3):239-243.JI Y, XIONG Q Y, WEI Y N, et al. Establishment of an indirect immunofluorescence assay for detection of adherence of Mycoplasma hyorhinis to host cells[J]. Chinese Journal of Zoonoses, 2014, 30(3):239-243.(in Chinese)
[22]	AYE R, MWIRIGI M K, FREY J, et al. Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells[J]. BMC Vet Res, 2015, 11:27.
[23]	辛九庆.牛传染性胸膜肺炎诊断技术与分子流行病学研究[D].长春:吉林农业大学, 2007.XIN J Q. Study of diagnosis technique and molecular epidemiology of contagious bovine pleuropneumonia[D]. Changchun:Jilin Agricultural University, 2007.(in Chinese)
[24]	HIRAI Y, KANATANI T, ONO M, et al. An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears[J]. Microbiol Immunol, 1991, 35(10):831-839.
[25]	NASEEM S, MEENS J, JORES J, et al. Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony type[J]. Vet Microbiol, 2010, 142(3-4):285-292.
[26]	GAO Y L, HUANG X X, ZHU Y B, et al. A brief review of monoclonal antibody technology and its representative applications in immunoassays[J]. J Immunoassay Immunochem, 2018, 39(4):351-364.
[27]	RODRÍGUEZ F, FERNÁNDEZ A, BALL H J. Detection of Mycoplasma mycoides subspecies mycoides by growth-inhibition using monoclonal antibodies[J]. Res Vet Sci, 1997, 63(1):91-92.
[28]	HWANG Y S, PANANGALA V S, ROSSI C R, et al. Monoclonal antibodies that recognize specific antigens of Mycoplasma gallisepticum and M. synoviae[J]. Avian Dis, 1989, 33(1):42-52.
[29]	BROOKS C, FINLAY D, BLACKBURN P, et al. Detection of Mycoplasma mycoides subspecies mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA[J]. Res Vet Sci, 2009, 87(2):200-203.
[30]	RODRIGUEZ F, BALL H J, FINLAY D, et al. Detection of Mycoplasma mycoides subspecies mycoides by monoclonal antibody-based sandwich ELISA[J]. Vet Microbiol, 1996, 51(1-2):69-76.
[31]	LE GOFF C, THIAUCOURT F. A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP)[J]. Vet Microbiol, 1998, 60(2-4):179-191.

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

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