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"

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