Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (19): 3936-3944.doi: 10.3864/j.issn.0578-1752.2024.19.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Preparation of Monoclonal Antibody Against African Swine Fever Virus p54 Protein and Identification of Its Epitope

FENG ChunYing(), ZHANG ZhaoXia, LIU YunFei, HUANG Li(), WENG ChangJiang   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory for Animal Disease Control and Prevention/Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin 150069
  • Received:2023-11-30 Accepted:2024-08-26 Online:2024-10-01 Published:2024-10-09
  • Contact: HUANG Li

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Abstract:

【Objective】The aim of this study was to obtain soluble African swine fever virus (ASFV) p54 protein and anti-p54 monoclonal antibodies (mAb), along with the identification of their recognized epitopes, so as to lay a foundation for the study of the structure and function of p54 protein and the development of serological diagnostic reagents. 【Method】To prepare mAb against ASFV p54 protein, the prokaryotic recombinant expression plasmid pET-21a-E183L was constructed and transformed into E.coli BL21 (DE3) cells. The recombinant protein p54 was purified by Ni-affinity chromatography and gel filtration, five weeks old BALB/c mice were immunized with the purified p54 recombinant protein. The immunization protocol involved three rounds at two-week intervals, starting with an emulsion of the antigen and an equal volume of Freund’s complete adjuvant, followed by the same antigen emulsion with an equal volume of Freund’s incomplete adjuvant for the subsequent immunization. After three immunizations, the blood of mice was collected, and the serum antibody titers were detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titers were selected for a booster immunization. Spleen cells from the mice were fused with SP2/0 cells three days later. The positive hybridoma cells were screened by indirect ELISA with the recombinant p54 protein. The specificity of the selected mAb was further confirmed via Western blot and indirect immunofluorescence assay (IFA). The isotype of the mAb was detected using a subclass identification kit. Subsequently, the p54 protein was systematically truncated and expressed as GST fusion proteins to identify the antigen epitopes recognized by the mAbs by Western blot. 【Result】 The constructed prokaryotic expression plasmid pET-21a-E183L (54-183 aa) was transformed into BL21 (DE3) cells. After induction with IPTG, the p54 recombinant protein was expressed in the supernatant in a soluble form with a molecular weight of about 17 kDa. Immunization of mice with purified p54 recombinant protein results in a serum titer of 1﹕409 600 seven days after the third immunization. Cell fusion was successfully performed. After four rounds of subcloning, a hybridoma cell line named 5B11 that could stably secrete mAb against p54 protein was obtained. The ascites fluid was prepared and the mAbs were purified. Western blot and IFA assay results showed that 5B11 could specifically recognize p54 protein expressed in HEK293T cells and ASFV-infected porcine alveolar macrophages (PAMs). The mAb subclass identification showed that 5B11 was of IgG1 heavy chain and κ light chain. The epitope sequence recognized by 5B11 was 80VTPQPGTSKPA90.【Conclusion】In this study, the recombinant protein ASFV p54 with amino acid 54-183 was successfully expressed in a soluble form in the prokaryotic system. The development of anti-p54 mAbs and the identification of their recognized epitopes have expanded understanding of p54 protein epitopes and provided the basic materials for the serological detection of ASFV.

Key words: African swine fever virus, p54 protein, monoclonal antibody, epitope

Fig. 1

Construction and identification of pET-21a-E183L recombinant vector A. PCR amplification results of E183L gene; B. Results of double enzyme digestion for identification of pET-21a-E183L. M: DL 2000 DNA Marker; 1: E183L gene; 2: Negative control; 3: Nde I and Xho I digestion of pET-21a-E183L; 4: Nde I and Xho I digestion of pET-21a"

Fig. 2

SDS-PAGE detection and Western blot identification results of His-p54 recombinant protein M: Protein molecular weight standard; 1: pET-21a-E183L/BL21 recombinant bacteria lysate without IPTG induction; 2: pET-21a- E183L/BL21 recombinant bacteria lysate induced by IPTG; 3: pET-21a-E183L/BL21 recombinant bacteria lysate supernatant induced by IPTG; 4: pET-21a-E183L/BL21 recombinant bacteria lysate precipitation induced by IPTG; 5: SDS-PAGE identification of His-p54 recombinant protein; 6: Western blot identification of His-p54 recombinant protein"

Fig. 3

Serum titer of immunized mouse was determined by ELISA"

Fig. 4

SDS-PAGE analysis of purified 5B11 mAb M: Protein molecular weight standard; 1: Purified 5B11 mAb"

Fig. 5

Western blot analysis for the reactivity of anti-p54 mAb with p54 proteins A, B. Western blot detected the expression of Flag-p54 protein; C. Western blot detected the expression of p54 protein in PAMs infected by ASFV"

Fig. 6

IFA analysis for the reactivity of anti-p54 mAb with p54 proteins A. IFA detected the expression of Flag-p54 protein; B. IFA detected the expression of p54 protein in PAMs infected by ASFV"

Fig. 7

Subtype identification of mAb"

Fig. 8

Identification of antigenic epitopes recognized by mAb by Western blot"

[1]
魏天, 王成宇, 王凤杰, 李忠鹏, 张芳毓, 张守峰, 扈荣良, 吕礼良, 王永志. 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. 中国农业科学, 2022, 55(15):3062-3070. doi: 10.3864/j.issn.0578-1752.2022.15.016.
WEI T, WANG C Y, WANG F J, LI Z P, ZHANG F Y, ZHANG S F, HU R L, LÜ L L, WANG Y Z. Preparation of monoclonal antibodies against the p30 protein of African swine fever virus and its mapping of linear epitopes. Scientia Agricultura Sinica, 2022, 55(15): 3062-3070. doi: 10.3864/j.issn.0578-1752.2022.15.016.
[2]
DIXON L K, STAHL K, JORI F, VIAL L, PFEIFFER D U. African swine fever epidemiology and control. Annual Review of Animal Biosciences, 2020, 8: 221-246.

doi: 10.1146/annurev-animal-021419-083741 pmid: 31743062
[3]
GALINDO-CARDIEL I, BALLESTER M, SOLANES D, NOFRARÍAS M, LÓPEZ-SORIA S, ARGILAGUET J M, LACASTA A, ACCENSI F, RODRÍGUEZ F, SEGALÉS J. Standardization of pathological investigations in the framework of experimental ASFV infections. Virus Research, 2013, 173(1): 180-190.
[4]
SÁNCHEZ-CORDÓN P J, MONTOYA M, REIS A L, DIXON L K. African swine fever: A re-emerging viral disease threatening the global pig industry. The Veterinary Journal, 2018, 233: 41-48.
[5]
GALINDO I, CUESTA-GEIJO M A, HLAVOVA K, MUÑOZ- MORENO R, BARRADO-GIL L, DOMINGUEZ J, ALONSO C. African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis. Virus Research, 2015, 200: 45-55.

doi: 10.1016/j.virusres.2015.01.022 pmid: 25662020
[6]
PENRITH M L, BASTOS A D, ETTER E M C, BELTRÁN- ALCRUDO D. Epidemiology of African swine fever in Africa today: Sylvatic cycle versus socio-economic imperatives. Transboundary and Emerging Diseases, 2019, 66(2): 672-686.
[7]
GE S Q, LI J M, FAN X X, LIU F X, LI L, WANG Q H, REN W J, BAO J Y, LIU C J, WANG H, LIU Y T, ZHANG Y Q, XU T G, WU X D, WANG Z L. Molecular characterization of African swine fever virus, China, 2018. Emerging Infectious Diseases, 2018, 24(11): 2131-2133.
[8]
SALAS M L, ANDRÉS G. African swine fever virus morphogenesis. Virus Research, 2013, 173(1): 29-41.

doi: 10.1016/j.virusres.2012.09.016 pmid: 23059353
[9]
WANG N, ZHAO D M, WANG J L, ZHANG Y L, WANG M, GAO Y, LI F, WANG J F, BU Z G, RAO Z H, WANG X X. Architecture of African swine fever virus and implications for viral assembly. Science, 2019, 366(6465): 640-644.

doi: 10.1126/science.aaz1439 pmid: 31624094
[10]
ALEJO A, MATAMOROS T, GUERRA M, ANDRÉS G. A proteomic atlas of the African swine fever virus particle. Journal of Virology, 2018, 92(23): e01293-18.
[11]
KARGER A, PÉREZ-NÚÑEZ D, URQUIZA J, HINOJAR P, ALONSO C, FREITAS F, REVILLA Y, LE POTIER M F, MONTOYA M. An update on African swine fever virology. Viruses, 2019, 11(9): 864.
[12]
CHAPMAN D A G, DARBY A C, DA SILVA M, UPTON C, RADFORD A D, DIXON L K. Genomic analysis of highly virulent Georgia 2007/1 isolate of African swine fever virus. Emerging Infectious Diseases, 2011, 17(4): 599-605.
[13]
RODRIGUEZ F, LEY V, GÓMEZ-PUERTAS P, GARCÍA R, RODRIGUEZ J, ESCRIBANO J. The structural protein p54 is essential for African swine fever virus viability. Virus Research, 1996, 40(2): 161-167.

pmid: 8725112
[14]
李子萱, 张永红, 童津津, 崔德凤. 非洲猪瘟病毒结构蛋白p54的功能研究进展. 动物医学进展, 2023, 44(8): 72-75.
LI Z X, ZHANG Y H, TONG J J, CUI D F. Progress on functions of structurel protein p54 of African swine fever virus. Progress in Veterinary Medicine, 2023, 44(8): 72-75. (in Chinese)
[15]
RODRÍGUEZ J M, GARCÍA-ESCUDERO R, SALAS M L, ANDRÉS G. African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites. Journal of Virology, 2004, 78(8): 4299-4313.

pmid: 15047843
[16]
DIXON L, SÁNCHEZ-CORDÓN P, GALINDO I, ALONSO C. Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis. Viruses, 2017, 9(9): 241.
[17]
REIS A L, PARKHOUSE R M E, PENEDOS A R, MARTINS C, LEITÃO A. Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus. Journal of General Virology, 2007, 88(9): 2426-2434.
[18]
赵改红, 张涛清, 向志达, 张元峰, 李江南, 翁长江. 非洲猪瘟病毒p54蛋白原核表达及多克隆抗体的制备. 中国动物传染病学报, 2023, 31(1): 166-173.
ZHAO G H, ZHANG T Q, XIANG Z D, ZHANG Y F, LI J N, WENG C J. Prokaryotic expression of African swine fever virus p54 protein and preparation of rabbit anti-p54 polyclonal antibodies. Chinese Journal of Animal Infectious Diseases, 2023, 31(1): 166-173. (in Chinese)
[19]
许文杰, 薛梦迪, 张元峰, 李长尧, 黄丽, 熊涛, 翁长江. 非洲猪瘟病毒pl215L蛋白单克隆抗体的制备及其反应性研究. 中国预防兽医学报, 2021, 43(4): 405-410.
XU W J, XUE M D, ZHANG Y F, LI C Y, HUANG L, XIONG T, WENG C J. Study on the preparation and reactivity of monoclonal antibody against pl215L protein of African swine fever virus. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(4): 405-410. (in Chinese)
[20]
仇华吉. 非洲猪瘟对我国养猪业的影响与防控建议. 中国兽药杂志, 2018, 52(11): 1-4.
QIU H J. Impacts of African swine fever on the Chinese pig industry and suggestions for the prevention and control of the disease. Chinese Journal of Veterinary Drug, 2018, 52(11): 1-4. (in Chinese)
[21]
赵启祖, 王琴. 非洲猪瘟紧急预防控制技术需求. 中国兽药杂志, 2018, 52(10): 1-4.
ZHAO Q Z, WANG Q. Technical requirements in African swine fever emergency prevention and control. Chinese Journal of Veterinary Drug, 2018, 52(10): 1-4. (in Chinese)
[22]
WU K K, LIU J M, WANG L X, FAN S Q, LI Z Y, LI Y W, YI L, DING H X, ZHAO M Q, CHEN J D. Current state of global African swine fever vaccine development under the prevalence and transmission of ASF in China. Vaccines, 2020, 8(3): 531.
[23]
孙爱军, 王芮, 朱潇静, 孟泽锟, 刘思源, 张帅, 靳家鑫, 张改平, 庄国庆. 非洲猪瘟相关检测及猪场生物安全防控研究进展. 中国兽医学报, 2021, 41(5): 1023-1030.
SUN A J, WANG R, ZHU X J, MENG Z K, LIU S Y, ZHANG S, JIN J X, ZHANG G P, ZHUANG G Q. Review on African swine fever-associated detection and pig farm biosecurity control measurement development. Chinese Journal of Veterinary Science, 2021, 41(5): 1023-1030. (in Chinese)
[24]
WANG A P, JIANG M, LIU H L, LIU Y K, ZHOU J M, CHEN Y M, DING P Y, WANG Y W, PANG W S, QI Y H, ZHANG G P. Development and characterization of monoclonal antibodies against the N-terminal domain of African swine fever virus structural protein, p54. International Journal of Biological Macromolecules, 2021, 180: 203-211.

doi: 10.1016/j.ijbiomac.2021.03.059 pmid: 33737177
[25]
CAO Y L, HAN D M, ZHANG Y J, ZHANG K, DU N N, TONG W, LI G X, ZHENG H, LIU C L, GAO F, TONG G Z. Identification of one novel epitope targeting p54 protein of African swine fever virus using monoclonal antibody and development of a capable ELISA. Research in Veterinary Science, 2021, 141: 19-25.

doi: 10.1016/j.rvsc.2021.10.008 pmid: 34638027
[26]
NEILAN J G, ZSAK L, LU Z, BURRAGE T G, KUTISH G F, ROCK D L. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection. Virology, 2004, 319(2): 337-342.

pmid: 14980493
[27]
梁云浩, 曹琛福, 陶虹, 吕建强, 唐勇. 非洲猪瘟病毒P54蛋白的真核表达及间接ELISA检测方法的建立. 中国兽医科学, 2014, 44(4): 373-378.
LIANG Y H, CAO C F, TAO H, LÜ J Q, TANG Y. Eukaryotic expression of African swine fever virus P54 protein and development of an indirect ELISA for detection of antibody against ASFV. Chinese Veterinary Science, 2014, 44(4): 373-378. (in Chinese)
[28]
GAO Y N, XIA T T, BAI J, ZHANG L J, ZHENG H X, JIANG P. Preparation of monoclonal antibodies against the viral p54 protein and a blocking ELISA for detection of the antibody against African swine fever virus. Viruses, 2022, 14(11): 2335.
[29]
戴建华, 王豪伟, 张紫菡. 基于p54的非洲猪瘟抗体胶体金检测方法建立. 中国动物传染病学报, 2021, 29(6): 50-54.
DAI J H, WANG H W, ZHANG Z H. Development of P54 based colloidal gold detection method for detection of African swine fever antibodies. Chinese Journal of Animal Infectious Diseases, 2021, 29(6): 50-54. (in Chinese)
[30]
苗朝悦, 杜乐, 王佳琦, 陈梓杰, 黄靖倍, 陈且昕, 邹沛璇, 韩笑, 张纯. 重组蛋白质在大肠杆菌体系中的可溶性表达策略. 中国生物工程杂志, 2023, 43(9): 33-45.
MIAO Z Y, DU L, WANG J Q, CHEN Z J, HUANG J B, CHEN J X, ZOU P X, HAN X, ZHANG C. Soluble expression strategies for production of recombinant proteins in Escherichia coli. China Biotechnology, 2023, 43(9): 33-45. (in Chinese)
[31]
郑南南, 东梦珂, 吴宏举, 侯浩宇, 陈寅龙, 李潮, 赵慧君, 张改平, 杜永坤. 非洲猪瘟病毒p54蛋白的截短表达及其单克隆抗体的制备. 中国兽医学报, 2022, 42(11): 2139-2144.
ZHENG N N, DONG M K, WU H J, HOU H Y, CHEN Y L, LI C, ZHAO H J, ZHANG G P, DU Y K. Prokaryotic expression of African swine fever virus p54 protein and preparation of monoclonal antibodies against p54 protein. Chinese Journal of Veterinary Science, 2022, 42(11): 2139-2144. (in Chinese)
[32]
伦玉潇, 李桂梅, 单虎. 非洲猪瘟病毒截短p54蛋白的原核表达与纯化. 动物医学进展, 2023, 44(2): 1-7.
LUN Y X, LI G M, SHAN H. Prokaryotic expression and purification of p54 truncated protein of African swine fever virus. Progress in Veterinary Medicine, 2023, 44(2): 1-7. (in Chinese)
[33]
PETROVAN V, MURGIA M V, WU P, LOWE A D, JIA W, ROWLAND R R R. Epitope mapping of African swine fever virus (ASFV) structural protein, p54. Virus Research, 2020, 279: 197871.
[34]
ZHAO H J, WANG G J, DONG H X, WU S Y, DU Y K, WAN B, JI P C, WU Y N, JIANG D W, ZHUANG G Q, DUAN H, ZHANG G P, ZHANG A K. Identification of a linear B cell epitope on p54 of African swine fever virus using nanobodies as a novel tool. Microbiology Spectrum, 2023, 11(3): e0336222.
[35]
齐艳丽, 刘桃雪, 于海深, 张超, 鲁维飞, 王江, 褚贝贝, 张改平. 非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定. 畜牧兽医学报, 2023, 54: 281-292.

doi: 10.11843/j.issn.0366-6964.2023.01.026
QI Y L, LIU T X, YU H S, ZHANG C, LU W F, WANG J, CHU B B, ZHANG G P. Preparation of the monoclonal antibody against the african swine fever virus p54 protein and identification of the antigenic epitope. Acta Veterinaria et Zootechnica Sinica, 2023, 54: 281-292. (in Chinese)

doi: 10.11843/j.issn.0366-6964.2023.01.026
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