China Animal Husbandry and Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (2): 781-791.doi: 10.16431/j.cnki.1671-7236.2025.02.028

• Preventive Veterinary Medicine • Previous Articles    

Preparation of Monoclonal Antibody Against African Swine Fever Virus p22 Protein and Identification of the Antigenic Epitope

WANG Xiaoge1,2, SI Xuanying1,2, YAN Zhiwei1,2, WANG Fei3, YOU Longqi1,2, LIU Geng1,2, CAI Mao1,2, LIANG Juncheng1,2, LIANG Yuxiu1,2, DU Yongkun1,2, ZHANG Gaiping1,2   

  1. 1. International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
    2. Henan Provincial Animal Biological Products Engineering Research Center, Zhengzhou 450046, China;
    3. Animal Disease Prevention and Control Center, Agriculture and Rural Bureau, Fugu County, Shaanxi Province, Yulin 719499, China
  • Received:2024-06-18 Published:2025-01-24

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Abstract: 【Objective】 The purpose of this study was to express and obtain recombinant protein p22 of African swine fever virus (ASFV) using the prokaryotic expression system,and further prepare and identify the monoclonal antibody against recombinant protein p22. 【Method】 PCR was used to amplify the ASFV p22 protein coding gene and construct the recombinant expression plasmid pET-28a(+)-p22，and induced to express p22 protein by IPTG.The purified recombinant protein was immunized to mice,and the spleen cells of immunized mice were fused with SP2/0 myeloma cells to prepare monoclonal antibody.The specificity of monoclonal antibody was determined by Western blotting,and the titer of ascites was determined by ELISA. Online software was used to predict the distribution of p22 protein epitopes and synthesize overlapping peptides,and the epitopes recognized by antibodies were identified by Dot blotting. 【Result】 In this study,the prokaryotic expression plasmid of p22 recombinant protein was successfully constructed,and the recombinant protein p22 expressed in the prokaryotic system was obtained with a molecular mass of about 22 ku.After immunizing mice with purified p22 protein as immunogen,4 monoclonal hybridoma cell lines were successfully screened，which was 1G3D7G11,2G5H6H8,6D10G7D6 and 8F6F8B9,and the ascites titer reached 1∶500 000.Western blotting results showed that 4 monoclonal antibodies could react specifically with p22 protein.The identification results of monoclonal subtypes showed that the heavy chains of monoclonal antibodies of 1G3D7G11,2G5H6H8 and 6D10G7D6 were IgG1,the heavy chain of monoclonal antibody of 8F6F8B9 was IgG2a,and the light chains of the 4 monoclonal antibodies were Kappa.Dot blotting test results showed that the epitopes of the recognition antigen of monoclonal antibodies 1G3D7G11 and 2G5H6H8 were located at amino acid 58-89.The epitope of monoclonal antibody 8F6F8B9 was located at amino acid 126-150. 【Conclusion】 In this study,4 monoclonal antibodies against ASFV p22 protein were successfully prepared,and the B cell epitope interval recognized by the monoclonal antibody was preliminarily identified.The results provided a reference for the study of biological function of p22 protein and serological detection of ASFV.

Key words: African swine fever virus; p22 protein; monoclonal antibody; epitope

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