弓形虫GRA1蛋白表达及间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2025.07.031

中国畜牧兽医 ›› 2025, Vol. 52 ›› Issue (7): 3308-3320.doi: 10.16431/j.cnki.1671-7236.2025.07.031

弓形虫GRA1蛋白表达及间接ELISA检测方法的建立

张翩^1,2, 陈晶¹, 张晓晓^1,2, 麦小鹏³, 唐科³, 向华¹, 王刚¹, 罗胜军¹, 马惠海⁴, 袁子国², 王晓虎¹

1. 广东省农业科学院动物卫生研究所, 农业农村部禽流感等家禽重大疾病防控重点实验室, 广东省畜禽疫病防治研究重点实验室, 广东省动物疫病野外科学观测研究站, 广州 510640;
2. 华南农业大学兽医学院, 广州 510642;
3. 广州市正安食品有限公司, 广州 511458;
4. 吉林省农业科学院(中国农业科技东北创新中心) 畜牧兽医研究所, 长春 136100

收稿日期:2024-10-17 出版日期:2025-07-05 发布日期:2025-07-01
通讯作者: 袁子国, 王晓虎 E-mail:ziguoyuan@scau.edu.cn;wangxiaohu2020@163.com
作者简介:张翩，E-mail：1737380188@qq.com；陈晶，E-mail：chenjing19827@163.com。
基金资助:
广州市科技计划项目(2023B04J0137、2023E04J1256)；2024年乡村振兴战略专项省级组织实施项目(2024-440000-900300-0114)；南沙预制菜进出口贸易关键共性技术与标准体系制定及示范推广应用服务项目；广东省畜禽疫病防治研究重点实验室项目(2023B1212060040)

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

ZHANG Pian^1,2, CHEN Jing¹, ZHANG Xiaoxiao^1,2, MAI Xiaopeng³, TANG Ke³, XIANG Hua¹, WANG Gang¹, LUO Shengjun¹, MA Huihai⁴, YUAN Ziguo², WANG Xiaohu¹

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

摘要/Abstract

摘要： 【目的】通过原核系统表达弓形虫GRA1蛋白，并建立其抗体间接ELISA(iELISA)检测方法，为猪弓形虫抗体检测提供材料。【方法】构建重组质粒pET-28a-GRA1，利用大肠杆菌表达系统表达GRA1蛋白，并用His镍柱进行GRA1蛋白纯化，经Western blotting鉴定其免疫原性。以纯化的GRA1蛋白作为抗原包被于固相载体，并用棋盘法确定抗原最佳包被浓度和待测血清稀释度。在此基础上，优化抗原包被条件、封闭条件、待测血清稀释度和酶标二抗稀释度。通过测定阴性血清D_{450 nm}值确定所建立方法的临界值，并进行特异性、敏感性和重复性试验，同时测定该方法与改良凝集试验(MAT)对实际样品检测的符合率。【结果】 SDS-PAGE结果显示，GRA1蛋白以可溶性形式表达，蛋白分子质量约为25 ku。Western blotting显示GRA1蛋白与猪弓形虫阳性血清发生特异性反应。优化后的ELISA反应条件为：抗原最佳包被浓度为5 μg/mL，血清稀释度为1∶100，抗原最佳包被条件为37 ℃包被2 h，最佳封闭条件为5%脱脂奶粉37 ℃作用2 h，待检血清作用时间为60 min，酶标二抗稀释度为1∶20 000，TMB作用时间为15 min。该检测方法的临界值为0.277。该方法敏感性良好，血清按1∶6 400稀释后仍可检测到阳性；批内和批间重复性变异系数均＜10%，重复性好；与猪带绦虫、旋毛虫、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒阳性血清均无交叉反应，具有良好的特异性。利用该方法和MAT方法对100份临床猪血清进行检测，总符合率为94.0%。【结论】本研究建立了一种快速有效的检测猪弓形虫抗体的间接ELISA方法，有助于猪感染弓形虫病的诊断和预防。

关键词: 猪; 弓形虫; GRA1; 原核表达; 间接ELISA

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 D_{450 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

中图分类号:

张翩, 陈晶, 张晓晓, 麦小鹏, 唐科, 向华, 王刚, 罗胜军, 马惠海, 袁子国, 王晓虎. 弓形虫GRA1蛋白表达及间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2025, 52(7): 3308-3320.

ZHANG Pian, CHEN Jing, ZHANG Xiaoxiao, MAI Xiaopeng, TANG Ke, XIANG Hua, WANG Gang, LUO Shengjun, MA Huihai, YUAN Ziguo, WANG Xiaohu. Expression of GRA1 Protein of Toxoplasma gondii and Establishment of an Indirect ELISA Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3308-3320.

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弓形虫GRA1蛋白表达及间接ELISA检测方法的建立

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

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