基于黏膜sIgA抗体的非洲猪瘟病毒感染早期血清学检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.01.028

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (1): 300-310.doi: 10.11843/j.issn.0366-6964.2024.01.028

基于黏膜sIgA抗体的非洲猪瘟病毒感染早期血清学检测方法的建立

白昀^1,3, 谢青云^1,3, 欧阳伟^1,3, 甘源^1,3, 袁厅^1,3, 赵东明², 步志高², 邵国青^1,3, 冯志新^1,3*

1. 江苏省农业科学院兽医研究所农业农村部兽用生物制品工程重点实验室, 南京 210014;
2. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室, 哈尔滨 150069;
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2023-02-06 出版日期:2024-01-23 发布日期:2024-01-24
通讯作者: 冯志新,主要从事动物疫病疫苗与诊断研究,E-mail:fzxjaas@163.com
作者简介:白昀(1980-),男,陕西华阴人,副研究员,硕士,主要从事抗体诊断试剂研究,E-mail:sunnybaiy@sina.com,Tel:025-84390880;谢青云(1990-),女,助理研究员,博士,主要从事抗体诊断试剂研究,E-mail:xqy816626@163.com,Tel:025-84390880。白昀和谢青云为同等贡献作者。
基金资助:
国家重点研发计划政府间国际科技创新合作重点专项（2019YFE0107300）；江苏省农业科技自主创新项目（CX（20）2035）

Establishment of a Serological Method for Early Detection of African Swine Fever Virus Infection Based on Mucosal sIgA Antibody

BAI Yun^1,3, XIE Qingyun^1,3, OUYANG Wei^1,3, GAN Yuan^1,3, YUAN Ting^1,3, ZHAO Dongming², BU Zhigao², SHAO Guoqing^1,3, FENG Zhixin^1,3*

1. Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
3. Guo Tai(Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2023-02-06 Online:2024-01-23 Published:2024-01-24

摘要/Abstract

摘要： 目前的非洲猪瘟病毒（ASFV）血清学检测方法存在因采血引起交叉感染风险、抗体转阳滞后影响检测敏感性等问题，因此建立一种无创采样且可实现早期诊断的血清学方法对于在临床上ASFV感染的诊断与监测有重要意义。本研究以人工合成的方式构建了pFastbac1-P30-His 重组表达载体，利用杆状病毒-昆虫细胞真核表达系统表达ASFV重组P30蛋白（SUMO-P30），用Ni柱亲和纯化后作为包被抗原，经过一系列条件优化，建立一种以猪口腔液中黏膜sIgA抗体为靶标的ASFV抗体间接ELISA方法。结果显示，真核重组表达的P30蛋白（SUMO-P30）约为 47 ku，Western blot 鉴定显示其具有良好的反应原性；经优化确定了ELISA最佳反应条件：包被量为1.0 μg·mL^－1，5%脱脂乳为最佳样品稀释液，与待检口腔液的最佳混合比例为2∶8，样品反应时间为120 min，鼠抗猪 IgA-HRP 最佳稀释度为1∶5 000，反应时间为60 min，最佳显色时间为15 min。该方法检测ASFV感染口腔液抗体效价可达1∶32，与猪瘟病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒黏膜抗体阳性口腔液均无交叉反应，具有良好的敏感性和特异性。利用该方法和ASFV商品化ELISA血清抗体检测试剂盒，分别检测感染ASFV强毒株或人工致弱株后同一头猪不同时间点的口腔液和血清样品，口腔液中黏膜sIgA抗体在感染后3~5 d S/P值就显著提升，而血清抗体在监测期内未检测到转阳。检测人工感染或同圈感染自然变异株后不同时间点的口腔液和血清样品，本研究建立的方法与商品化非洲猪瘟病毒血清抗体检测试剂盒检测的阳性符合率为100%，阴性符合率为37.5%，总符合率为61.5%。综上，本研究建立了一种无需采血，以口腔液为样品的ASFV黏膜sIgA抗体ELSIA检测方法，可实现ASFV感染的无创、早期、敏感诊断，为ASFV的监测和防控提供新的技术支持和补充。

关键词: 非洲猪瘟, sIgA抗体, ELISA, 早期诊断

Abstract: At present, there is a risk of cross-infection caused by blood collection in the commonly used serological detection methods of African swine fever virus (ASFV) antibodies, and the serological detection sensitivity is affected by the relatively delayed serological conversion of serum antibodies. Therefore, it is important to establish a non-invasive sampling serological method that can realize early diagnosis and monitoring of ASFV infection. In this study, pFastbac1-P30-His recombinant expression vector was constructed by artificial synthesis. The recombinant ASFV P30 protein (SUMO-P30) was expressed by baculovirus-insect cell eukaryotic expression system, purified by Ni column affinity and used as the coating antigen. After a series of conditions optimization, an indirect ELISA method of ASFV antibody targeting the mucosal sIgA antibody in porcine oral fluid was established. The results showed that the eukaryotic recombinant P30 protein (SUMO-P30) was about 47 ku, and Western blot analysis showed that it had good reactivity; The optimal reaction condition of ELISA was determined after optimization: the coating amount was 1.0 μg·mL^－1, 5% skimmed milk was the best sample dilution, the best mixing ratio with the oral liquid to be tested was 2∶8, the sample reaction time was 120 min, the best dilution of rat anti-pig IgA-HRP was 1∶5 000, the reaction time was 60 min, and the best color development time was 15 min. The antibody titer of ASFV infected oral fluid detected by this method could reach 1∶32, and there was no cross reaction with CSFV, PRV, PRRSV mucosal antibody positive oral fluid, which indicated its good sensitivity and specificity. By using this method and the ASFV commercialized ELISA serum antibody detection kit, the oral fluid and serum samples of the same pig infected with ASFV virulent or artificially attenuated strains were detected at different time points. The mucosal sIgA antibody in the oral fluid showed a significant increase in S/P values 3-5 days after infection, while serum antibodies were not detected to be positive during the monitoring period. Oral fluid and serum samples from challenged or indirectly infected piglet with natural variant ASFV strains were detected at different time points post infection. The positive coincidence rate of this method with the commercial ASFV antibody detection kit was 100%, the negative coincidence rate was 37.5%, and the total coincidence rate was 61.5%. In conclusion, this study has established an ELSIA detection method of ASFV mucosal antibody with oral fluid as sample without blood collection, which can realize non-invasive early diagnosis of ASFV infection. This method provides new technical support and supplement for ASFV monitoring, prevention and control.

Key words: African swine fever, sIgA antibody, ELISA, early diagnosis

中图分类号:

S852.4

白昀, 谢青云, 欧阳伟, 甘源, 袁厅, 赵东明, 步志高, 邵国青, 冯志新. 基于黏膜sIgA抗体的非洲猪瘟病毒感染早期血清学检测方法的建立[J]. 畜牧兽医学报, 2024, 55(1): 300-310.

BAI Yun, XIE Qingyun, OUYANG Wei, GAN Yuan, YUAN Ting, ZHAO Dongming, BU Zhigao, SHAO Guoqing, FENG Zhixin. Establishment of a Serological Method for Early Detection of African Swine Fever Virus Infection Based on Mucosal sIgA Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 300-310.

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基于黏膜sIgA抗体的非洲猪瘟病毒感染早期血清学检测方法的建立

Establishment of a Serological Method for Early Detection of African Swine Fever Virus Infection Based on Mucosal sIgA Antibody

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