A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.12.027

摘要/Abstract

摘要： 本研究旨在制备A型口蹄疫病毒(FMDV-A)多表位纳米融合蛋白并建立磁微粒CLIA抗体检测方法，为FMDV的临床监测和防控提供技术支撑。以表达的FMDV-A型多表位融合蛋白及其多克隆抗体为基础建立FMDV-A型磁微粒CLIA抗体检测方法，优化反应条件，并与商品化试剂盒同时进行临床样品的检测，比较两种方法的符合率。结果显示：本研究最终成功制备了高可溶性FMDV-A型多表位纳米融合蛋白及其特异性多克隆抗体，多克隆抗体效价为1∶5 120，并根据竞争法免疫原理建立了FMDV-A型磁微粒CLIA抗体检测方法。SA-Beads最佳浓度为0.5 mg·mL^-1，生物素标记的FMDV-A型重组蛋白抗原最佳工作浓度为5 μg·L^-1，AE标记的多克隆抗体最佳工作浓度为2 μg·L^-1；最佳上样体积为10 μL，最佳反应时间为15 min。敏感性试验结果表明，该方法的敏感性为97.47%，敏感性好。特异性试验结果表明，与其他相似病原没有交叉反应，特异性强。重复性试验结果说明变异系数CV值均小于5%，具有良好的稳定性和重复性。临床试验结果表明，与中国农科院兰州兽医研究所LPB-ELISA方法比较，总符合率为96.76%，为国内FMDV-A的研究与快速诊断建立了技术基础。本研究成功制备FMDV-A型毒株的VP1多表位融合纳米颗粒蛋白，并获得其高效价多克隆抗体，应用该蛋白和多克隆抗体建立了FMDV-A型磁微粒CLIA抗体检测方法。

关键词: A型口蹄疫病毒, 铁蛋白, CLIA, 磁微粒化学发光, 纳米颗粒

Abstract: This experiment was conducted to prepare a multi-epitope nanofusion protein of foot-and-mouth disease virus serotype A (FMDV-A) and to establish a magnetic particle CLIA antibody detection method, which was to provide technical support for clinical immunization monitoring, prevention and control of FMDV. In this study, a magnetic particle chemiluminescent immunoassay (CLIA) for detecting antibodies against foot-and-mouth disease virus serotype A (FMDV-A) was established based on the expressed multi-epitope fusion protein of FMDV-A and its polyclonal antibody. Reaction conditions were optimized, and clinical samples were simultaneously tested using this method and commercial kits to compare the coincidence rate between the two methods. Finally, a highly soluble FMDV-A multi-epitope nanofusion protein and specific polyclonal antibody with a titer of 1∶5 120 were successfully prepared. A magnetic particle CLIA for FMDV-A antibodies detection was established based on the principle of competitive immunoassay. The optimal concentration of streptavidin magnetic beads (SA-Beads) was 0.5 mg·mL^-1, the optimal concentration of biotin-labeled recombinant FMDV-A protein antigen was 5 μg·L^-1, and the optimal concentration of acridinium ester (AE)-labeled polyclonal antibody was 2 μg·L^-1. The optimal sample volume was 10 μL, and the optimal reaction time was 15 minutes. The sensitivity test results show that the sensitivity of this method is 97.47%, and the sensitivity is good. No cross-reactivity was observed with other clinically similar pathogens, demonstrating strong specificity. The results of repeatability test demonstrated that coefficient of variation (CV) values were less than 5%, which had good stability and repeatability The results of the clinical trials indicated that the overall coincidence rate was 96.76% when compared with the LPB-ELISA method developed by Lanzhou Veterinary Research Institute. This research has established a technical foundation for the research and rapid diagnosis on type A foot-and-mouth disease in China. This study successfully prepared a VP1 multi epitope fusion nanoparticle protein of FMDV-A strain and obtained its high titer polyclonal antibody. The protein and polyclonal antibody were used to establish a detection method for FMDV-A magnetic particle CLIA antibody.

Key words: foot-and-mouth disease virus type A, ferritin, CLIA, magnetic particles chemiluminescence, nanoparticle

中图分类号:

S855.3

郑雪莹, 王培, 孙伟珊, 赵浩军, 殷雨晴, 李佳, 程汝佳, 刘海莹, 王大猛, 穆伟, 李达, 陈锡俊, 郑博君, 梅力. A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立[J]. 畜牧兽医学报, 2025, 56(12): 6257-6270.

ZHENG Xueying, WANG Pei, SUN Weishan, ZHAO Haojun, YIN Yuqing, LI Jia, CHENG Rujia, LIU Haiying, WANG Dameng, MU Wei, LI Da, CHEN Xijun, ZHENG Bojun, MEI Li. Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6257-6270.

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