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中国农学通报

中国农学通报 ›› 2025, Vol. 41 ›› Issue (33): 157-164.doi: 10.11924/j.issn.1000-6850.casb2025-0241

• 畜牧·动物医学·蚕·蜂 • 上一篇    

基于非洲猪瘟病毒P72重组蛋白的间接ELISA方法的初步建立

向国庆(), 邓天博, 宋帅, 杨燕秋, 陈玉婷, 陈美霞, 吴国景, 郑博彬, 牛瑞辉, 温肖会(), 罗胜军()   

  1. 广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站，广州 510640
  • 收稿日期:2025-03-19 修回日期:2025-08-26 出版日期:2025-11-25 发布日期:2025-12-01
  • 通讯作者:
    温肖会，女，1982年出生，河南安阳人，研究员，硕士，主要从事动物疫病净化防控研究。通信地址：510640 广东省广州市天河区五山白石岗街1号，E-mail：；
    罗胜军，男，1974年出生，安徽望江人，研究员，硕士，主要从事动物疫病防控与监测研究。通信地址：510640 广东省广州市天河区五山白石岗街1号，E-mail：。
  • 作者简介:

    向国庆，男，1998年出生，湖南怀化人，硕士，研究方向：动物疫病与防控。通信地址：510640 广东省农业科学院动物卫生研究所，E-mail：。

  • 基金资助:
    广东省科技计划项目“广东省畜禽疫病防治研究重点实验室”(2023B1212060040); 广东省农业科学院动物卫生研究所青年科技人才培育项目“猪重要病原管式化学发光免疫分析检测试剂盒研发”(PY2024010)

Preliminary Establishment of Indirect ELISA Method Based on African Swine Fever Virus P72 Recombinant Protein

XIANG Guoqing(), DENG Tianbo, SONG Shuai, YANG Yanqiu, CHEN Yuting, CHEN Meixia, WU Guojing, ZHENG Bobin, NIU Ruihui, WEN Xiaohui(), LUO Shengjun()   

  1. Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Guangdong Province Key Laboratory of Livestock Disease Prevention/Guangdong Provincial Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology, Ministry of Agriculture and Rural Affairs, Guangzhou 510640
  • Received:2025-03-19 Revised:2025-08-26 Published:2025-11-25 Online:2025-12-01

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摘要：

本研究旨在建立一种高特异性、高灵敏度且快速的间接ELISA方法，用于临床非洲猪瘟病毒(ASFV)抗体的高效检测。以重组质粒pET-28a-P72为表达载体，经IPTG诱导（终浓度0.5 mmol/L，37℃诱导4 h）和镍亲和纯化，获得高纯度重组蛋白P72（纯度>90%），通过Western blot验证其与ASFV阳性血清的反应原性（条带清晰，分子量约35 kDa，与阴性血清无交叉反应）。将其作为包被抗原，通过各项条件优化，初步建立非洲猪瘟病毒P72蛋白抗体间接ELISA方法。以该重组P72蛋白为包被抗原，通过棋盘滴定法优化关键参数，初步建立ASFV抗体间接ELISA检测方法。结果显示，优化后的方法学参数为：抗原包被浓度5 μg/mL、4℃封闭12 h；血清稀释度1:160、37℃反应30 min；酶标二抗稀释度1:1000、37℃反应30 min；TMB底物37℃反应10 min。判定标准基于40份ASFV阴性血清（健康猪血清）检测结果确定：OD450值≥0.69为阳性，OD450值≤0.57为阴性，OD450值在两者之间则判定为可疑。方法学评估表明：该方法与猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)等6种病原阳性血清均无交叉反应（特异性100%）；灵敏度可达1:1600；批内变异系数(CV)为3.2%~8.5%，批间CV为6.8%~12.3%，均小于15%，呈现出良好的准确度和可靠性。成功建立非洲猪瘟病毒P72蛋白抗体间接ELISA方法，可用于ASFV抗体的检测，并为疫情监测预警提供实用化技术支持。

关键词: 非洲猪瘟病毒, 重组P72蛋白, 原核表达, 间接ELISA

Abstract:

This study aimed to develop a rapid indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against African swine fever virus (ASFV) in clinical application, and the recombinant P72 protein was obtained using the recombinant plasmid pET-28a-P72 through IPTG induction and nickel affinity purification. Its reactivity was confirmed, and the purified protein was used as the coating antigen. After optimizing key parameters, an indirect ELISA method for detecting antibodies against ASFV P72 protein was preliminarily established. The ASFV P72 protein was successfully expressed and specifically reacted with ASFV-positive serum antibodies. The optimized assay conditions were as follows: coating antigen concentration at 5 μg/mL, serum dilution at 1:160, coating at 4℃ for 12 h, serum incubation time of 30 min, HRP-labeled secondary antibody dilution at 1:1000, secondary antibody incubation time of 30 min, and substrate reaction time of 10 min. The cut-off values were defined as follows: OD450≥0.69 was considered positive, OD450≤0.57 was negative, and values between these thresholds were considered suspicious. No cross-reactivity was observed with antisera against six pathogens including classical swine fever virus (CSFV). The sensitivity reached 1:1600, and both intra- and inter-batch coefficients of variation were less than 15%, demonstrating high accuracy and reliability. An indirect ELISA method for detecting antibodies against ASFV P72 protein was successfully established. This assay can be used for ASFV antibody detection and provides technical support for epidemic monitoring and early warning.

Key words: African swine fever virus, recombinant P72 protein, prokaryotic expression, indirect ELISA