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

摘要/Abstract

摘要： 【目的】制备非洲猪瘟病毒（African swine fever virus，ASFV） p30蛋白单克隆抗体，为ASFV检测方法的研究提供重要试验材料。【方法】应用大肠杆菌表达重组ASFV p30蛋白，并用该蛋白免疫6~8周龄的雌性BALB/c小鼠，应用细胞融合技术将免疫小鼠脾细胞与SP2/0细胞融合；通过间接ELISA方法筛选分泌p30蛋白特异性抗体的阳性杂交瘤细胞，腹腔接种小鼠制备腹水抗体；应用ELISA方法鉴定单克隆抗体的类/亚类，通过Western blotting和间接免疫荧光试验（IFA）鉴定单克隆抗体与p30-N端（1―105位氨基酸）和p30-C端（100―194位氨基酸）区域的反应活性；针对抗体识别的p30蛋白区域合成不同肽段，通过ELISA和斑点免疫印迹法鉴定抗体识别的抗原表位。应用间接ELISA方法分析单克隆抗体与ASFV p72蛋白、猪圆环病毒2型（PCV2）衣壳（Cap）蛋白、猪流行性腹泻病毒（PEDV） S蛋白的交叉反应性，以及ASFV抗血清对单克隆抗体抗原结合活性的阻断作用。【结果】获得了2株杂交瘤细胞（C7和G10），其分泌的单克隆抗体（C7和G10）都属于IgG1亚类和κ型（IgG1κ），杂交瘤细胞培养上清和腹水内C7和G10的抗体效价分别为1：1 280、1：640与1：10⁷、1：10⁶；2株抗体均与p30-C端（100―194位氨基酸）区域特异性结合，并识别同一个抗原表位¹¹⁵CTSSFETLFEQEPSSEVPKD¹³⁴；2株抗体与ASFV p72蛋白、PCV2 Cap蛋白及PEDV S蛋白无交叉反应；ASFV抗血清能有效阻断2株单克隆抗体与p30蛋白结合。【结论】获得2株分泌p30单克隆抗体的杂交瘤细胞株，鉴定了单克隆抗体的抗原识别表位，丰富了p30蛋白的抗原表位信息，为ASFV致病机制的进一步研究提供支撑。

关键词: 非洲猪瘟病毒(ASFV); p30蛋白; 单克隆抗体; 表位

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

中图分类号:

S852.65⁺1

马天天, 张亚楠, 冯亚文, 岳怀宁, 王亚文, 苏恺, 袁晨, 逯纪成, 孙泰然, 薛文阁, 宋勤叶. 非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2023, 50(7): 2832-2842.

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.

参考文献

[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.