口蹄疫病毒VP1蛋白的原核表达及间接ELISA抗体检测方法的建立  

Prokaryotic Expression of VP1 Protein of Foot-and-Mouth Disease Virus and Development of an Indirect ELISA for Antibody Detection

作　　者：张芳源 杜文琪 杨大为 李桂梅 单虎 ZHANG Fangyuan;DU Wenqi;YANG Dawei;LI Guimei;SHAN Hu(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Novel Veterinary Pharmacy Innovation Center of Shandong Province,Qingdao 266109,China;Research Center for Engineering Technology in Veterinary Medicine and Veterinary Diagnostic Reagent of Shandong Province,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,青岛266109 [2]山东省新兽药创制协同创新中心,青岛266109 [3]山东省兽药诊断试剂工程技术研究中心,青岛266109

出　　处：《中国动物传染病学报》2025年第1期82-90,共9页Chinese Journal of Animal Infectious Diseases

基　　金：科技创新2030—重大项目课题(2021ZD0113802)。

摘　　要：为建立口蹄疫病毒(FMDV)快速、高效的血清学检测方法,根据GenBank收录的FMDV-O型国内流行株VP1基因(GenBank登录号:JN998085.1),构建重组质粒pCold TF-VP1,通过原核表达系统获得VP1蛋白。通过SDS-PAGE和Western blot鉴定蛋白表达和反应原性后,将纯化后的蛋白作为抗原,建立FMDV抗体间接ELISA方法。结果显示,重组质粒成功转入大肠杆菌BL21(DE3)感受态细胞中,并以可溶性蛋白形式表达。建立的FMDV抗体间接ELISA方法其抗原包被最佳浓度0.5μg/mL,使用1%BSA溶液封闭效果最佳,血清最佳稀释度1∶600,酶标二抗最佳工作浓度1∶8000,且具有良好的特异性、灵敏性和重复性,与商品化试剂盒比较,符合率为90.2%。本试验在大肠杆菌中成功表达并纯化了FMDV的VP1蛋白,并建立了FMDV抗体间接ELISA方法,为口蹄疫的监测、诊断及试剂盒的开发提供了参考。In order to develop a rapid and accurate serological method for detection of Foot-and-mouth disease virus(FMDV)antibodies,the recombinant plasmid pCold TF-VP1 containing the VP1 gene(GenBank accession number:JN998085.1)of FMDV-O type was constructed for prokaryotic expression.The expressed VP1 protein was confirmed in SDS-PAGE and Western blot and purified as an antigen for development of a indirect ELISA.The results showed that the VP1 gene was expressed as a soluble protein in E.coli BL21(DE3)cells.The indirect ELISA was developed and optimized including antigen coating concentration at 0.5μg/mL,1%BSA blocking solution,serum dilution at 1:600,and HRP-labeled secondary antibody dilution at 1:8000.The method showed good specificity,high sensitivity and good reproducibility.In addition,the indirect ELISA had the consistent rate at 90.2%as compared with the commercial kit.In summary,the VP1 protein of FMDV was expressed in E.coli and purified for development of an indirect ELISA.This work might provide a foundation for the monitoring,diagnosis and development of a diagnostic kit for FMDV.

关 键 词：口蹄疫病毒 VP1蛋白 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]