China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (7): 3308-3320.doi: 10.16431/j.cnki.1671-7236.2025.07.031

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Expression of GRA1 Protein of Toxoplasma gondii and Establishment of an Indirect ELISA Detection Method

ZHANG Pian1,2, CHEN Jing1, ZHANG Xiaoxiao1,2, MAI Xiaopeng3, TANG Ke3, XIANG Hua1, WANG Gang1, LUO Shengjun1, MA Huihai4, YUAN Ziguo2, WANG Xiaohu1   

  1. 1. Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Livestock Disease Prevention, Guangdong Provincial Observation and Research Station for Animal Disease, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
    2. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
    3. Guangzhou Zhengan Company Food Co., Ltd., Guangzhou 511458, China;
    4. Livestock and Veterinary Research Institute, Jilin Academy of Agricultural Sciences (Northeast Innovation Center for Agricultural Science and Technology in China), Changchun 136100, China
  • Received:2024-10-17 Online:2025-07-05 Published:2025-07-01

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Abstract: 【Objective】 This study was conducted to express GRA1 protein of Toxoplasma gondii in prokaryotic expression system,and develop an indirect ELISA (iELISA) method for rapid detection of Toxoplasma gondii antibody,and provide material for serological investigation of Toxoplasma gondii. 【Method】 The recombinant plasmid pET-28a-GRA1 was constructed,the GRA1 protein was expressed by Escherichia coli expression system,and the protein was purified with His nickel column.The immunogenicity of the protein was identified by Western blotting.The purified GRA1 protein was coated on the solid-phase carrier as antigen,and the optimal antigen coating concentration and serum dilution were determined by checkerboard titration method.On this basis,the antigen coating conditions,blocking conditions,serum dilution to be tested and enzyme-labeled secondary antibody dilution were optimized.The cutoff value of the established method was determined by measuring the D450 nm value of negative sera,and the specificity,sensitivity and reproducibility tests were carried out.At the same time,the coincidence rate between the method and the modified agglutination test (MAT) for the detection of actual samples was determined. 【Result】 The results of SDS-PAGE showed that the GRA1 protein was expressed in soluble form,and the relative molecular weight of the protein was 25 ku.Western blotting result showed that the GRA1 protein reacted specifically with the porcine positive serum of Toxoplasma gondii.The optimized ELISA reaction conditions were:The optimal antigen encapsulation concentration was 5 μg/mL,with a serum dilution of 1∶100,antigen encapsulation was performed at 37 ℃ for 2 h,and the optimal containment condition was 5% skimmed milk powder at 37 ℃ for 2 h,and the action time of the serum to be tested was 60 min,the dilution of enzyme labeled secondary antibody was 1∶20 000,and the action time of TMB was 15 min.The cutoff value of this detection method was 0.277.This method had good sensitivity,positive results could still be detected when the serum was diluted at a ratio of 1∶6 400.The coefficients of variation of intra- and inter-assay repeatability were both less than 10%,indicating good repeatability.It had no cross-reaction with positive sera of Taenia solium,Trichinella spiralis,Porcine pseudorabies virus or Porcine reproductive and respiratory syndrome virus,and had good specificity.One hundred clinical porcine sera were tested using this method and the MAT method,and the total coincidence rate was 94.0%. 【Conclusion】 In this study,a rapid and effective indirect ELISA method for detecting antibodies of Toxoplasma gondii in pigs was established,which was helpful for the diagnosis and prevention of Toxoplasma gondii infection in pigs.

Key words: pig; Toxoplasma gondii; GRA1; prokaryotic expression; indirect ELISA

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