中国兽医科学2025，Vol.55Issue(5)：608-615,8.DOI:10.16656/j.issn.1673-4696.2025.0075
布鲁氏菌BP26蛋白原核表达及双抗原夹心ELISA方法的建立
Prokaryotic expression of BP26 protein in Brucella and establishment of a double-antigen sandwich ELISA method
摘要
Abstract
To establish an ELISA method for differential detection of Brucella serum antibodies,for distinguishing BP26 gene deletion vaccine immunity.Based on the reference sequence of Brucella M5-90 strain published by NCBI,this study predicted the homology,transmembrane region,and antigenicity of BP26,and constructed two vectors,pET-28a-BP26 and pMAL-c2x-BP26.The protein was expressed in E.coli and purified by affinity chromatography.Western-blot was used to analyze the reactivity of the target protein.His-BP26 was used as the coating antigen,and HRP labeled MBP-BP26 was used as the detection antigen.After optimization,a double antigen sandwich ELISA method was established.According to sta-tistical analysis,the critical value of the double antigen sandwich ELISA is 0.35.After testing,the method had no cross-reactivity with foot-and-mouth disease virus-O,lumpy skin disease virus,bovine viral diarrhea virus antibody positive serum and rakabane disease virus polyantibody,with a sensitivity of 1∶640;the intra-batch and inter-batch coefficient of variation was less than 10％.Using this method,40 known positive and negative sheep serum samples were tested,with 8 positive and 32 negative results.This indicates that the ELISA method established in this study has strong specificity,high sensitivity,good reproducibility,and can distinguish BP26 gene deletion vaccine immunity,which is used for sero-logical investigation and clinical diagnosis of Brucellosis.关键词
布鲁氏菌/BP26/原核表达/双抗原夹心ELISAKey words
Brucella/BP26/prokaryotic expression/double antigen sandwich ELISA分类
农业科技引用本文复制引用
席韬,徐继英,田宏,郑海学,罗俊聪,朱昱茜,林永玉,陈婕,张帆,石鑫泰,何印娣,李帅鹏..布鲁氏菌BP26蛋白原核表达及双抗原夹心ELISA方法的建立[J].中国兽医科学,2025,55(5):608-615,8.基金项目
家畜重要疫病感染与免疫防控的基础理论研究(22ZD6NA001) （22ZD6NA001）
中央高校基本科研业务费专项资金资助(学科交叉创新团队建设项目)(lzujbky-2022-ct02) （学科交叉创新团队建设项目）
中国农业科学院创新计划项目(CAAS-CSLPDCP-2023002) （CAAS-CSLPDCP-2023002）
国家生猪技术创新中心项目(NCTIP-XD/C03) （NCTIP-XD/C03）
家畜重要疫病诊断研制与产业化项目(22ZD6NA012) （22ZD6NA012）
农业技术产业体系建设项目(CARS-35) （CARS-35）
"十四五"广东省揭榜挂帅项目(2023SDZG02) （2023SDZG02）
第三批甘肃省拔尖领军人才培养项目 （）
甘肃省临床兽医实验教学示范中心项目 （）
