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

Abstract

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

CLC Number:

S852.659.4

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.

References 28

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

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