基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2020.12.022

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (12): 3133-3140.doi: 10.11843/j.issn.0366-6964.2020.12.022

基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

易华山¹, 赵瑶¹, 马鲜平¹, 李欢¹, 夏宇¹, 朱文青¹, 刘倩如¹, 陈思倩¹, 曹慧贞¹, 姚婷¹, 高丽旭¹, 张金阳², 杨发挥³, 魏晓蓉⁴, 李前勇^1*, 谢远兵^5*

1. 西南大学动物医学院, 重庆 402460;
2. 昆明理工大学生命科学与技术学院, 昆明 650500;
3. 重庆市荣昌区农业技术推广站, 重庆 402460;
4. 重庆纳比威特检测技术服务有限公司, 重庆 402460;
5. 重庆市永川区动物疫病预防控制中心, 重庆 402160

收稿日期:2020-06-10 出版日期:2020-12-25 发布日期:2020-12-23
通讯作者: 李前勇,主要从事临床兽医学研究,E-mail:1411642119@qq.com;谢远兵,主要从事动物流行病学研究,E-mail:342562770@qq.com
作者简介:易华山(1980-),男,甘肃临夏人,博士,主要从事兽医临床感染与免疫研究,E-mail:dyxyihuashan@swu.edu.cn;赵瑶(1994-),女,云南泸西人,硕士生,主要从事临床兽医感染与免疫研究,E-mail:790542142@qq.com。
基金资助:
重庆市基础研究与前沿探索专项项目基金（cstc2018jcyjAX0615）；中央高校基本科研业务专项基金（XDJK2018C059；XDJK2018C060）；国家重点研发计划项目（2018YFD0501705）

Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies

YI Huashan¹, ZHAO Yao¹, MA Xianping¹, LI Huan¹, XIA Yu¹, ZHU Wenqing¹, LIU Qianru¹, CHEN Siqian¹, CAO Huizhen¹, YAO Ting¹, GAO Lixu¹, ZHANG Jinyang², YANG Fahui³, WEI Xiaorong⁴, LI Qianyong^1*, XIE Yuanbing^5*

1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
2. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China;
3. Chongqing Rongchang Agricultural Technical Extension Station, Chongqing 402460, China;
4. Chongqing Nabiweit Testing Technology Service Co. LTD, Chongqing 402460, China;
5. Yongchuan Center for Animal Disease Prevention and Control, Chongqing 402160, China

Received:2020-06-10 Online:2020-12-25 Published:2020-12-23

摘要/Abstract

摘要： 旨在建立蓝舌病病毒（BTV）血清学ELISA抗体检测方法，本研究以原核表达并纯化的BTV NS4重组蛋白为包被抗原，通过反应条件优化，建立了一种BTV重组NS4蛋白的间接ELISA抗体检测方法。SDS-PAGE结果显示，获得大小约52 ku的NS4重组融合蛋白，主要在上清中存在，Western blot显示，纯化后的重组蛋白具有良好的抗原性。通过方阵试验进行了ELISA反应条件优化，确定了重组蛋白抗原最佳包被量为3.0 μg·孔^-1；血清最佳稀释倍数为1：200，酶标二抗最佳工作浓度为1：4 000，临界值分别为0.29和0.35。上述以NS4蛋白作为包被抗原建立的BTV抗体间接ELISA方法检测敏感性可达1：1 600；批内和批间重复性变异系数均小于10%；检测76份重庆地区牛群血清样品，阳性符合率为98%，阴性符合率为100%。本研究建立的间接ELISA方法为临床BTV血清抗体检测及BTV血清流行病学调查奠定了基础。

关键词: 蓝舌病病毒, NS4基因, 原核表达, 间接ELISA

Abstract: This study was conducted to establish an indirect ELISA method for the detection serological antibody of bluetongue virus (BTV). The purified BTV recombinant NS4 protein obtained from the prokaryotic express system was used as the coated antigen, and then an indirect ELISA antibody detection method of BTV was developed by optimizing the reaction conditions. SDS-PAGE results showed that the recombinant NS4 protein with a size of about 52 kDa was obtained, which mainly existed in the supernatant. Western blot results showed that the purified recombinant NS4 protein had good antigenicity. The ELISA reaction conditions were optimized by the square matrix test. The optimal coating amount of recombinant NS4 protein antigen was determined to be 3.0 μg per well, and the optimal dilution ratio of serum to be tested was 1:200, and the optimal dilution concentration of HRP-labeled rabbit anti-cow IgG secondary antibody was 1:4 000, and the critical values were 0.29 and 0.35, respectively. The detection sensitivity of the BTV antibody was up to 1:1 600. The intra-assay repeatability and the inter-assay repeatability coefficient of variation were less than 10%. The positive coincidence ratio and negative coincidence ratio were 98% and 100% respectively. The indirect ELISA method established in this study laid a foundation for clinical serum antibody detection and serum epidemiological investigation of BTV.

Key words: bluetongue virus, NS4 gene, prokaryotic expression, indirect ELISA

中图分类号:

S852.659.4

易华山, 赵瑶, 马鲜平, 李欢, 夏宇, 朱文青, 刘倩如, 陈思倩, 曹慧贞, 姚婷, 高丽旭, 张金阳, 杨发挥, 魏晓蓉, 李前勇, 谢远兵. 基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2020, 51(12): 3133-3140.

YI Huashan, ZHAO Yao, MA Xianping, LI Huan, XIA Yu, ZHU Wenqing, LIU Qianru, CHEN Siqian, CAO Huizhen, YAO Ting, GAO Lixu, ZHANG Jinyang, YANG Fahui, WEI Xiaorong, LI Qianyong, XIE Yuanbing. Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3133-3140.

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基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies

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