China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (1): 270-279.doi: 10.16431/j.cnki.1671-7236.2023.01.027

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method

ZHANG Kai1, ZHANG Chunping2, GE Shengqiang3, SUN Chunxi1, CHENG Jie1, FU Chunyu1, WANG Gang1, NIU Xing4, PENG Jun1   

  1. 1. Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271000, China;
    2. Weifang Center for Animal Disease Prevention and Control in Shandong Province, Weifang 261000, China;
    3. China Animal Health and Epidemiology Center, Qingdao 266032, China;
    4. College of Modern Agriculture, Linyi Science and Technology Vocational College, Linyi 276000, China
  • Received:2022-06-21 Online:2023-01-05 Published:2023-01-04

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Abstract: 【Objective】 This study was aimed to express and purify the structural protein p22 of African swine fever virus (ASFV), and use it as a coating antigen to establish an indirect ELISA method for detecting ASFV antibodies and diagnosis of African swine fever.【Method】 The truncated KP177R gene encoding ASFV p22 protein (amino acids 24-145) was cloned into prokaryotic expression vector pET-32a(+) to construct the expressing plasmid pET-32a-p22.The recombinant plasmid was transformed into Escherichia coli (E.coli) BL21(DE3) competent cells and then induced by 0.1 mmol/L IPTG for 5 h.The corresponding p22 protein was purified by affinity chromatography on Ni ion and identified by Western blotting.BALB/c mice were inoculated with the purified p22 protein to prepare antiserum.Meanwhile, HEK293T cells were transfected with the eukaryotic expression plasmid containing the p22 full-length gene fragment (pCAGGS-EGFP-fp22) and used as antigenic matrix.Indirect immunofluorescence assay (IFA) was used to identify the reactivity of the antiserum.Based on the optimized parameters including the coating antigen concentration, serum dilution, blocking condition, reaction time of the first antibody, and working concentration of the enzyme-conjugated secondary antibody, an indirect ELISA method was established for ASFV antibody detection, and the clinical porcine serum samples were detected using this indirect ELISA method.【Result】 The truncated ASFV p22 protein was expressed in E.coli, and the protein concentration was 0.85 mg/100 g of bacteria.The prokaryotic p22 protein showed good immunogenicity, and the titer of the corresponding antiserum could reach 1:12 800.The optimized parameters of the ELISA (p22-ELISA)were as follows:The concentration of coating antigen was 0.125 μg/well, the dilution of serum was 1:800, and the dilution of the secondary antibody was 1:10 000.The cut-off value of p22-ELISA was 0.384.There was no cross reaction with CSFV, PRRSV, PCV2 and PRV antibody positive porcine serum, indicating good specificity.The coefficients of variation intra- and inter-batch were lower than 10%, indicating good repeatability.The p22-ELISA was used to detect 263 clinical pig serum samples, and the coincidence rate with thecommercial ASFV ELISA kit was 97.7%.【Conclusion】 This study established an indirect ELISA method for ASFV antibody detection based on the prokaryotic truncated ASFV p22 protein, the method had strong specificity, high sensitivity and good repeatability, and provided technical storage for the diagnosis and epidemiological investigation of ASFV infection.

Key words: African swine fever virus(ASFV); p22 protein; prokaryotic expression; indirect ELISA; antibody

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