非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2023.07.023

Abstract

Abstract: 【Objective】 To provide important experimental materials for the research of African swine fever virus (ASFV) detection method, the monoclonal antibody against p30 protein of ASFV was prepared.【Method】 Recombinant ASFV p30 protein was expressed by Escherichia coli and used to immunize female BALB/c mice aged 6-8 weeks.The splenocytes of the immunized mice were fused with SP2/0 cells by cell fusion technique.The positive hybridoma cells secreting specific antibody against p30 protein were screened by indirect ELISA, and the hybridoma cells were intraperitoneally inoculated into mice to produce ascites antibodies.The class/subclass of the monoclonal antibodies were identified by ELISA, and the reactivity of the monoclonal antibody with p30-N terminal (1-105 amino acids) and p30-C terminal (100-194 amino acids) of p30 protein were identified by Western blotting and indirect immunofluorescence assay (IFA).Different peptides were synthesized for the p30 protein region recognized by the antibody, and the epitopes recognized by antibodies were identified by ELISA and Dot blotting.Indirect ELISA was used to analyze the cross-reactivity of the monoclonal antibodies with ASFV p72 protein, Porcine circovirus type 2 (PCV2) capsid (Cap) protein, Porcine epidemic diarrhea virus (PEDV) S protein, and the blocking effect of ASFV antiserum on the antigen binding activity of monoclonal antibodies.【Result】 Two hybridoma cells (C7 and G10) were obtained, and the monoclonal antibodies (C7 and G10) secreted by the two cell lines belonged to the IgG1 subclass and κ type (IgG1κ).The titers of C7 and G10 in hybridoma cell culture supernatant and ascites were 1:1 280, 1:640, and 1:10⁷, 1:10⁶, respectively.The two monoclonal antibodies were specifically bound to the p30-C terminal (100-194 amino acids) region, and recognized the same epitope ¹¹⁵CTSSFETLFEQEPSSEVPKD¹³⁴.There was no cross reaction with ASFV p72, PCV2 Cap and PEDV S proteins, and anti-ASFV positive serum could effectively block the two monoclonal antibodies from binding to p30 protein.【Conclusion】 Two hybridoma cell lines secreting p30 monoclonal antibody were obtained, and the epitopes recognized by monoclonal antibodies were identified, which enriched the information of the epitopes of p30 protein, and laid an foundation for further research on the pathogenesis of ASFV.

Key words: African swine fever virus (ASFV); p30 protein; monoclonal antibody; epitope

CLC Number:

S852.65⁺1

MA Tiantian, ZHANG Yanan, FENG Yawen, YUE Huaining, WANG Yawen, SU Kai, YUAN Chen, LU Jicheng, SUN Tairan, XUE Wenge, SONG Qinye. Preparation and Identification of Monoclonal Antibody Against African Swine Fever Virus p30 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2832-2842.

References

[1] BLOME S, FRANZKE K, BEER M.African swine fever-A review of current knowledge[J].Virus Research, 2020, 287:198099.
[2] GALINDO I, ALONSO C.African swine fever virus:A review[J].Viruses, 2017, 9(5):103-113.
[3] 王清华, 任炜杰, 包静月, 等.我国首例非洲猪瘟的确诊[J].中国动物检疫, 2018, 35(9):1-4. WANG Q H, REN W J, BAO J Y, et al.The first outbreak of African swine fever was confirmed in China[J].China Animal Health inspection, 2018, 35(9):1-4.(in Chinese)
[4] JIA N, OU Y, PEJSAK Z, et al.Roles of African swine fever virus structural proteins in viral infection[J].Journal of Veterinary Research, 2017, 61(2):135-143.
[5] BOSCH-CAMOS L, LOPEZ E, RODRIGUEZ F.African swine fever vaccines:A promising work still in progress[J].Porcine Health Management, 2020, 6:17.
[6] TEKLUE T, SUN Y, ABID M, et al.Current status and evolving approaches to African swine fever vaccine development[J].Transboundary and Emerging Diseases, 2020, 67(2):529-542.
[7] 张文燕, 陈立功, 腾召剑, 等.基于非洲猪瘟病毒p30蛋白的间接ELISA抗体检测方法的建立[J].中国兽医学报, 2022, 42(7):1320-1326. ZHANG W Y, CHEN L G, TENG Z J, et al.Establishment of an indirect ELISA for detection of antibodies against African swine fever virus p30 protein[J].Chinese Journal of Veterinary Science, 2022, 42(7):1320-1326.(in Chinese)
[8] GAUDREAULT N N, MADDEN D W, WISLSON W C, et al.African swine fever virus:An emerging DNA Arbovirus[J]. Frontiers in Veterinary Science, 2020, 7:215.
[9] 黄霞, 康喜龙, 孟闯, 等.非洲猪瘟病毒及其疫苗研究进展[J].生物学杂志, 2021, 38(6):99-103. HUANG X, KANG X L, MENG C, et al.Research progress on African swine fever virus and its vaccines[J].Journal of Biology, 2021, 38(6):99-103.(in Chinese)
[10] ZHOU X, LI N, LUO Y, et al.Emergence of African swine fever in China, 2018[J].Transboundary and Emerging Diseases, 2018, 65(6):1482-1484.
[11] SUN E, ZHANG Z, WANG Z, et al.Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020[J].Science China Life Science, 2021, 64(5):752-765.
[12] WANG X, WANG X, ZHANG X, et al.Genetic characterization and variation of African swine fever virus China/GD/2019 strain in domestic pigs[J].Pathogens, 2022, 11(1):97.
[13] LACASTA A, BALLESTER M, MONTEAGUDO P L, et al.Expression library immunization can confer protection against lethal challenge with African swine fever virus[J].Journal of Virology, 2014, 88(22):13322-13332.
[14] NEILAN J G, ZSAK L, LU Z, et al.Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection[J].Virology, 2004, 319(2):337-342.
[15] NETHERTON C L, GOATLEY L C, REIS A L, et al.Identification and immunogenicity of African swine fever virus antigens[J].Frontiers in Immunology, 2019, 10:1318.
[16] ARGILAGUET J M, PEREZ-MARTIN E, GALLARDO C, et al.DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies[J].PLoS One, 2012, 7(9):e40942.
[17] OVIEDO J M, RODRIGUEZ F, GOMEZ-PUERTAS P, et al.High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents[J].Journal of Virological Methods, 1997, 64(1):27-35.
[18] PEREZ-FILGUEIRA D M, GONZALEZ-CAMACHO F, GALLARDO C, et al.Optimization and validation of recombinant serological tests for African swine fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[J].Journal of Clinical Microbiology, 2006, 44(9):3114-3121.
[19] YU X, ZHU X, CHEN X, et al.Establishment of a blocking ELISA detection method for against African swine fever virus p30 antibody[J]. Frontiers in Veterinary Science, 2021, 8:781373.
[20] 张路捷, 高雁怩, 夏婷婷, 等.非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定[J].畜牧与兽医, 2021, 53(6):111-115. ZHANG L J, GAO Y N, XIA T T, et al.Preparation and identification of monoclonal antibodies against p30 protein of the African swine fever virus[J].Animal Husbandry & Veterinary Medicine, 2021, 53(6):111-115. (in Chinese)
[21] ZHOU G, SHI Z, LUO J, et al.Preparation and epitope mapping of monoclonal antibodies against African swine fever virus P30 protein[J].Applied Microbiology and Biotechnology, 2022, 106(3):1199-1210.
[22] IMDHIVAS M, SEN S, BARMAN N, et al.Computational analysis of immunogenic epitopes in the p30 and p54 proteins of African swine fever virus[J].Journal of Biomolecular Structure & Dynamics, 2022, 1-10.doi:10.1080/07391102.2022.2123400.Online ahead of print.
[23] WU P, LOWE A D, RODRÍGUEZ Y Y, et al.Antigenic regions of African swine fever virus phosphoprotein P30[J].Transboundary and Emerging Diseases, 2020, doi:10.1111/tbed.13533.Online ahead of print.
[24] ZHOU J, NI Y, WANG D, et al.Development of a competitive enzyme-linked immunosorbent assay targeting the-p30 protein for detection of antibodies against African swine fever virus[J].Viruses, 2023, 15(1):154.
[25] 吕卉.抗非洲猪瘟病毒CD2v单克隆抗体的研制及p30抗原表位的鉴定[D].扬州:扬州大学, 2022. LYU H.Preparation of monoclonal antibody to CD2v of ASFV and a novel epitope identification of p30[D].Yangzhou:Yangzhou University, 2022.(in Chinese)
[26] 魏天, 王成宇, 王凤杰, 等.非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位[J].中国农业科学, 2022, 55(15):3062-3070. WEI T, WANG C Y, WANG F J, et al.Preparation of monoclonal antibodies against the p30 protein of African swine fever virus and its mapping of linear epitopes[J].Scientia Agricultural Sinica, 2022, 55(15):3062-3070.(in Chinese)
[27] 周改静, 罗俊聪, 石正旺, 等.非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立[J].畜牧兽医学报, 2022, 53(12):4337-4345. ZHOU G J, LUO J C, SHI Z W, et al.Preparation of monoclonal antibody against African swine fever virus p30 protein and establishment of a blocking ELlSA for detection of antibody against ASFV p30 protein[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(12):4337-4345.(in Chinese)
[28] YUAN F, PETROVAN V, GIMENEZ-LIROLA L G, et al.Development of a blocking enzyme-linked immunosorbent assay for detection of antibodies against African swine fever virus[J].Pathogens, 2021, 10(6):760.

Preparation and Identification of Monoclonal Antibody Against African Swine Fever Virus p30 Protein

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