非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2023.01.026

Abstract

Abstract: The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times’ subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all κ chains. The lowest titer of mAb was 1∶25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine.

Key words: African swine fever virus, p54 protein, prokaryotic expression, monoclonal antibodies, indirect ELISA, epitope identification

CLC Number:

S852.659.1

QI Yanli, LIU Taoxue, YU Haishen, ZHANG Chao, LU Weifei, WANG Jiang, CHU Beibei, ZHANG Gaiping. Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 281-292.

References 40

[1]	FIORI M S, SANNA D, SCARPA F, et al. A deeper insight into evolutionary patterns and phylogenetic history of ASFV epidemics in sardinia (Italy) through extensive genomic sequencing[J]. Viruses, 2021, 13(10):1994.
[2]	王凯. 非洲猪瘟病毒的传播途径与控制措施研究[J]. 中国畜牧业, 2022, 10(4):43-44.WANG K. Study on transmission route and control measures of ASFV[J]. China Animal Industry, 2022, 10(4):43-44. (in Chinese)
[3]	COSTARD S, WIELAND B, DE GLANVILLE W, et al. African swine fever:how can global spread be prevented?[J]. Philos Trans R Soc Lond B Biol Sci, 2009, 364(1530):2683-2696.
[4]	BORCA M V, RAMIREZ-MEDINA E, SILVA E, et al. ASFV-G-△I177L as an effective oral nasal vaccine against the Eurasia strain of Africa swine fever[J]. Viruses, 2021, 13(5):765.
[5]	ALEJO A, MATAMOROS T, GUERRA M, et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92(23):e01293-18.
[6]	GAUDREAULT N N, MADDEN D W, WILSON W C, et al. African swine fever virus:an emerging DNA arbovirus[J]. Front Vet Sci, 2020, 7:215.
[7]	SALAS M L, ANDRÉS G. African swine fever virus morphogenesis[J]. Virus Res, 2013, 173(1):29-41.
[8]	WANG N, ZHAO D M, WANG J L, et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366(6465):640-644.
[9]	DIXON L K, CHAPMAN D A G, NETHERTON C L, et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173(1):3-14.
[10]	安一娜, 杨静静, 高敏, 等. 非洲猪瘟病毒编码蛋白功能及疫苗研究进展[J]. 养殖与饲料, 2021, 20(10):107-114.AN Y N, YANG J J, GAO M, et al. Research progress on the function and vaccine of African Swine Fever virus coding protein[J]. Animals Breeding and Feed, 2021, 20(10):107-114. (in Chinese)
[11]	王涛, 孙元, 罗玉子, 等. 非洲猪瘟防控及疫苗研发:挑战与对策[J]. 生物工程学报, 2018, 34(12):1931-1942.WANG T, SUN Y, LUO Y Z, et al. Prevention, control and vaccine development of African swine fever:challenges and countermeasures[J]. Chinese Journal of Biotechnology, 2018, 34(12):1931-1942. (in Chinese)
[12]	孙茂文, 王涛, 孙元, 等. 非洲猪瘟病毒的免疫逃逸策略[J]. 微生物学报, 2021, 61(2):249-262.SUN M W, WANG T, SUN Y, et al. Immunoevasion strategies of African swine fever virus[J]. Acta Microbiologica Sinica, 2021, 61(2):249-262. (in Chinese)
[13]	SUN M W, YU S X, GE H L, et al. The A137R protein of African swine fever virus inhibits type I interferon production via the autophagy-mediated lysosomal degradation of TBK1[J]. J Virol, 2022, 96(9):e01957-21.
[14]	ALONSO C, MISKIN J, HERNEZ B, et al. African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein[J]. J Virol, 2001, 75(20):9819-9827.
[15]	GÓMEZ-PUERTAS P, RODRÍGUEZ F, OVIEDO J M, et al. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization[J]. J Virol, 1996, 70(8):5689-5694.
[16]	REIS A L, PARKHOUSE R M E, PENEDOS A R, et al. Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus[J]. J Gen Virol, 2007, 88(9):2426-2434.
[17]	雷有玲, 张文. 猪伪狂犬病毒野毒抗体间接ELISA检测方法的建立与应用[J]. 畜牧与兽医, 2014, 46(11):60-64.LEI Y L, ZHANG W. Establishment and application of indirect ELISA for detection of porcine pseudorabies virus wild virus antibody[J]. Animal Husbandry & Veterinary Medicine, 2014, 46(11):60-64. (in Chinese)
[18]	莫红芳, 揭凯, 陈鑫, 等. 猪塞尼卡谷病毒单克隆抗体的制备及鉴定[J]. 中国畜牧兽医, 2022, 49(1):307-317.MO H F, JIE K, CHEN X, et al. Preparation and identification of monoclonal antibodies against porcine Seneca valley virus[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(1):307-317. (in Chinese)
[19]	雷有玲, 李国泰, 鲍玉林, 等. 猪伪狂犬病毒gE蛋白单克隆抗体制备与鉴定[J]. 畜牧与兽医, 2018, 50(1):109-112.LEI Y L, LI G T, BAO Y L, et al. Preparation and identification of monoclonal antibodies against gE protein of pseudorabies virus[J]. Animal Husbandry & Veterinary Medicine, 2018, 50(1):109-112. (in Chinese)
[20]	郭瑞珍, 苏冰倩, 王棋, 等. 禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2021, 52(7):2000-2012.GUO R Z, SU B Q, WANG Q, et al. Preparation of the monoclonal antibody against the serotype 4 fowl adenovirus Fiber2 protein and identification of the antigenic epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7):2000-2012. (in Chinese)
[21]	孙娟, 王愉涵, 刘艳, 等. 抗原表位鉴定方法的研究进展[J]. 国外医学(医学地理分册), 2017, 38(3):291-295.SUN J, WANG Y H, LIU Y, et al. Research advances in the identification methods of antigen epitope[J]. Foreign Medical Sciences (Section of Medgeography), 2017, 38(3):291-295. (in Chinese)
[22]	李俊慧, 邵军军, 常惠芸. 抗原表位鉴定方法的研究进展[J]. 中国兽医科学, 2021, 51(6):678-683.LI J H, SHAO J J, CHANG H Y. Advance of antigenic epitope mapping methods[J]. Chinese Veterinary Science, 2021, 51(6):678-683. (in Chinese)
[23]	WANG Q, ZHOU L Y, WANG J, et al. African swine fever virus K205R induces ER stress and consequently activates autophagy and the NF-κB signaling pathway[J]. Viruses, 2022, 14(2):394.
[24]	CHEN W Y, ZHAO D M, HE X J, et al. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs[J]. Sci China Life Sci, 2020, 63(5):623-634.
[25]	GAUDREAULT N N, RICHT J A. Subunit vaccine approaches for African swine fever virus[J]. Vaccines (Basel), 2019, 7(2):56.
[26]	RUIZ-GONZALVO F, RODRÍGUEZ F, ESCRIBANO J M. Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus[J]. Virology, 1996, 218(1):285-289.
[27]	GÓMEZ-PUERTAS P, RODRÍGUEZ F, OVIEDO J M, et al. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response[J]. Virology, 1998, 243(2):461-471.
[28]	张皓淳. 非洲猪瘟病毒结构蛋白p54的优化表达及间接ELISA抗体检测方法的建立[J]. 国外畜牧学(猪与禽), 2022, 42(2):35-42.ZHANG H C. Optimization of expression of structural protein p54 of African swine Fever virus and establishment of indirect ELISA antibody detection method[J]. Animal Science Abroad (Pigs and Poultry), 2022, 42(2):35-42. (in Chinese)
[29]	徐玲玉, 曹琛福, 李中圣, 等. 非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立[J]. 畜牧兽医学报, 2022, 53(3):813-821.XU L Y, CAO C F, LI Z S, et al. Preparation of monoclonal antibody against African swine fever virus p54 protein and development of blocking ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3):813-821. (in Chinese)
[30]	官丽娟, 邵钰, 任伟杰, 等. 非洲猪瘟病毒结构蛋白p54的原核表达与反应原性鉴定[J]. 中国动物检疫, 2019, 36(12):63-67.GUAN L J, SHAO Y, REN W J, et al. Prokaryotic expression and reactivity identification of structural protein p54 of African swine fever virus[J]. China Animal Health Inspection, 2019, 36(12):63-67. (in Chinese)
[31]	郑丁丁, 逄文强, 王衡, 等. 非洲猪瘟病毒p54蛋白单克隆抗体的制备与鉴定[J]. 病毒学报, 2020, 36(6):1143-1150.ZHENG D D, PANG W Q, WANG H, et al. Preparation and identification of monoclonal antibodies against the recombinant p54 protein of African swine fever virus[J]. Chinese Journal of Virology, 2020, 36(6):1143-1150. (in Chinese)
[32]	TESFAGABER W, WANG L L, TSEGAY G, et al. Characterization of anti-p54 monoclonal antibodies and their potential use for African swine fever virus diagnosis[J]. Pathogens, 2021, 10(2):178.
[33]	WANG A P, JIANG M, LIU H L, et al. Development and characterization of monoclonal antibodies against the N-terminal domain of African swine fever virus structural protein, p54[J]. Int J Biol Macromol, 2021, 180:203-211.
[34]	PETROVAN V, MURGIA M V, WU P, et al. Epitope mapping of African swine fever virus (ASFV) structural protein, p54[J]. Virus Res, 2020, 279:197871.
[35]	CAO Y L, HAN D M, ZHANG Y J, et al. Identification of one novel epitope targeting p54 protein of African swine fever virus using monoclonal antibody and development of a capable ELISA[J]. Res Vet Sci, 2021, 141:19-25.
[36]	曹琛福, 梁云浩, 花群俊, 等. 非洲猪瘟病毒p54蛋白抗原表位预测及鉴定[J]. 中国预防兽医学报, 2014, 36(11):848-851.CAO C F, LIANG Y H, HUA Q J, et al. B cell epitope prediction and identification on the p54 of African swine fever virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(11):848-851. (in Chinese)
[37]	CORRAL-LUGO A, LÓPEZ-SILES M, LÓPEZ D, et al. Identification and analysis of unstructured, linear B-cell epitopes in SARS-CoV-2 virion proteins for vaccine development[J]. Vaccines (Basel), 2020, 8(3):397.
[38]	栗宁, 周景明, 王爱萍. 非洲猪瘟病毒主要结构蛋白B细胞表位的研究现状[J]. 动物医学进展, 2022, 43(4):93-96.LI N, ZHOU J M, WANG A P. Research status on B cell epitopes of major structural proteins of African swine fever virus[J]. Progress in Veterinary Medicine, 2022, 43(4):93-96. (in Chinese)
[39]	杨文兵, 邹亚文, 蒋一凡, 等. 非洲猪瘟血清学诊断靶点的研究进展[J]. 畜牧兽医学报, 2021, 52(5):1208-1217.YANG W B, ZOU Y W, JIANG Y F, et al. Advances research on African swine fever serological diagnostic targets[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5):1208-1217. (in Chinese)
[40]	LIU Q, MA B T, QIAN N C, et al. Structure of the African swine fever virus major capsid protein p72[J]. Cell Res, 2019, 29(11):953-955.

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope

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