China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (10): 4440-4449.doi: 10.16431/j.cnki.1671-7236.2024.10.025

• Preventive Veterinary Medicine • Previous Articles    

Prokaryotic Expression of P54 Protein of African Swine Fever Virus and Establishment of an Indirect ELISA Detection Method

WANG Cheng1,2, XIA Yingju2, WANG Haidong1, LIU Yebing2   

  1. 1. Shanxi Agricultural University, Taigu 032699, China;
    2. China Institute of Veterinary Drug Control, Beijing 100081, China
  • Received:2024-01-22 Published:2024-09-30

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Abstract: 【Objective】 This study was aimed to establish an indirect ELISA method for detecting antibodies against African swine fever virus (ASFV) with good specificity and sensitivity.【Method】 According to the gene sequence of ASFV P54 protein, E.coli codon optimisation was carried out,and the optimised P54 sequence was cloned into the pET-28a(+) plasmid to construct the recombinant plasmid pET28a-P54,which was used to express the target proteins using the E.coli expression system.Protein purification was carried out with a His nickel column,and after identification by SDS-PAGE and Western blotting,the purified P54 protein was encapsulated as a detection antigen to establish an indirect ELISA method,which was validated for its critical value,specificity,sensitivity and reproducibility after optimisation of the reaction conditions such as encapsulation conditions,closure solution,closure time and serum incubation.【Result】 The results of SDS-PAGE showed that the molecular weight of P54 protein was 42 ku,and the purity was above 95%.The results of Western blotting showed that P54 protein could react specifically with ASFV positive serum.The optimized ELISA conditions were as follows:0.3125 μg/mL antigen was coated at 37 ℃ for 1 h,5% skimmed milk powder was blocked at 37 ℃ for 1 h,serum diluted at 1∶800 was incubated at 37 ℃ for 2 h,and enzyme-labeled secondary antibody diluted at 1∶15 000 was incubated at 37 ℃ for 1 h.The ELISA method only reacted specifically with ASFV positive sera,and did not cross-react with positive sera of Swine fever virus,Porcine parvovirus,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Bovine viral diarrhoea virus,Porcine pseudorabies virus and Encephalitis B virus.The sensitivity of the method was high,and the ASFV-positive serum was still significantly responsive when the dilution was 1∶3 200.The intra-batch and inter-batch duplicates were both＜10%.Compared with the commercial kits,the positive compliance rate was 95.45%,the negative compliance rate was 96.05%,and the total compliance rate was 95.83%.【Conclusion】 In this study,an indirect ELISA method using ASFV P54 protein as antigen was established.The method had good specificity and sensitivity,and could be used for serological detection of ASFV,providing more options for rapid diagnosis of African swine fever.

Key words: African swine fever virus (ASFV); indirect ELISA method; P54 protein; prokaryotic expression

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