基于GNS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2023.10.028

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4320-4326.doi: 10.11843/j.issn.0366-6964.2023.10.028

基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

何辰香¹, 高闪电^1*, 田占成¹, 独军政¹, 王锦明¹, 关贵全¹, 殷宏^1,2*

1. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室, 兰州 730046;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2023-01-11 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 高闪电,E-mail:gaoshandian@caas.cn;殷宏,主要从事兽医微生物及其分子生物学研究,E-mail:yinhong@caas.cn
作者简介:何辰香(1997-),女,甘肃陇南人,硕士,主要从事家畜分子病毒学研究;高闪电(1980-),男,河北石家庄人,副研究员,主要从事家畜分子病毒学研究。
基金资助:
甘肃省重点研发计划（22YF7NA030）；甘肃省创新引导计划-科技特派团专项（22CX8NA011）；兰州市科技计划项目（2021-1-12）；国家重点研发计划项目（2021YFD1800500）；甘肃省基础研究创新群体项目（22JR5RA024）；国家肉牛牦牛产业技术体系（NBCIS，CARS-37）；农业科技创新工程（CAAS-ASTIP-2016-LVRI）。

Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle

HE Chenxiang¹, GAO Shandian^1*, TIAN Zhancheng¹, DU Junzheng¹, WANG Jinming¹, GUAN Guiquan¹, YIN Hong^1,2*

1. State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2023-01-11 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

摘要： 旨在建立牛流行热病毒（BEFV）感染与疫苗免疫的抗体ELISA诊断方法。在证实BEFV G_NS截短体与BEFV感染血清特异性反应的基础上，利用原核表达系统表达纯化G_NS全长蛋白并以其作为包被抗原，优化反应条件，建立基于BEFV G_NS的鉴别病毒感染与疫苗免疫的BEFV间接ELISA方法。结果显示：在大肠杆菌中成功表达G_NS蛋白，主要以包涵体的形式存在，纯化获得73 ku的重组蛋白。Western blot结果显示，纯化后的重组蛋白与BEFV感染牛血清反应原性良好，且与BEF疫苗免疫牛血清未见反应。G_NS抗原最佳包被浓度为0.50 μg·mL^-1，血清最佳稀释度为1：20，酶标二抗最佳稀释度为1：4 000，阴阳性临界值为S/P值0.206 9。该方法与BVDV、FMDV以及IBRV阳性血清无交叉反应，特异性良好；批内和批间变异系数均小于10%，重复性良好，可用于BEFV感染与疫苗免疫牛的鉴别诊断。

关键词: 牛流行热, 牛流行热病毒, 牛流行热灭活疫苗, G_NS蛋白, 鉴别诊断ELISA

Abstract: This study focused on developing an antibody-detecting ELISA capable of distinguishing BEFV infected and vaccinated animals. Based on the identified specific reactivity between the truncated BEFV G_NS protein and sera from BEFV infected cattle, the full-length G_NS recombinant protein was expressed in prokaryotic system, purified and used for developing an antibody-detecting ELISA to distinguish BEFV infected and vaccinated animals under the optimized reaction conditions. The G_NS was expressed in Escherichia coli and deposited mainly in inclusion bodies. The purified recombinant protein had a molecular weight of 73 ku. Western blot demonstrated that sera from BEFV infected cattle but not sera from BEF vaccine immunized cattle reacted strongly with the purified G_NS protein. The optimal concentrations of plate-coating antigens and the dilution of sera were 0.50 μg·mL^-1 and 1:20, the HRP conjugated secondary antibody was determined to be 1:4 000, and the S/P ratio=0.206 9 was selected as the negative/positive cut-off value. Acceptable specificity and repeatability were confirmed by no cross reactivity with positive sera of BVDV, FMDV and IBRV as well as coefficient of variation less than 10% in intra-batch and inter-bath. The DIVA (differentiation of infected from vaccinated animals) ELISA was developed and can be used for distinguish BEFV infected and vaccinated cattle.

Key words: bovine ephemeral fever, bovine ephemeral fever virus, BEF inactivated vaccine, G_NS protein, differential diagnostic ELISA

中图分类号:

S852.659.5

何辰香, 高闪电, 田占成, 独军政, 王锦明, 关贵全, 殷宏. 基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立[J]. 畜牧兽医学报, 2023, 54(10): 4320-4326.

HE Chenxiang, GAO Shandian, TIAN Zhancheng, DU Junzheng, WANG Jinming, GUAN Guiquan, YIN Hong. Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4320-4326.

参考文献 17

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基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle

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基于GNS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

Development of a Differential Diagnostic ELISA Based on GNS Protein to Distinguish BEFV Infected and Vaccinated Cattle

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基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle