中国兽医科学2025，Vol.55Issue(1)：65-72,8.DOI:10.16656/j.issn.1673-4696.2024.0213
肠炎沙门菌fliC基因的原核表达及其间接ELISA方法的建立
Prokaryotic expression of fliC gene in Salmonella Enteritidis and establishment of its indirect ELISA method
摘要
Abstract
To establish an indirect ELISA method based on FliC of Salmonella Enteritidis,the truncated FliC of Salmonella Enteritidis was expressed in Escherichia coli BL21(DE3)and used as a coated antigen to establish an indirect ELISA method.The negative and positive thresholds were determined by using 52 clinical chicken serum samples.The specificity,sensitivity and repeatability were determined by using positive chicken serum samples.The consistency and clinical applicability were compared to the BioChek commercial kits by detecting 135 clinical serum samples.The results showed that the soluble FliC could specifically bind to H∶g antibodies,not react with H∶i and H∶m antibodies.For the indirect ELISA,the optimal mass concentration of coated FliC was 4 ng/μL,the optimal dilution of the test serum was 1∶40,and the optimal dilution of the HRP second antibody was 1∶2 000.The negative threshold of this method was 0.82,and the positive threshold was 0.94.Moreover,the specificity results showed that there was no cross-reaction with Salmonella Pullorum,Salmonella Typhimurium,Salmonella Mbannkar,Salmonella Thomson and Salmonella Kentucky,and 80×diluted positive serum could still be detected.The coefficient of variation(CV)for inter-assay and intra-assay repeated detection were 0.43％to 5.13％and 0.50％to 2.68％respectively.The coincidence rate of detection with Salmonella avian group D ELISA was 94.1％.In conclusion,an indirect ELISA method for the detection of Salmonella Enteritidis,was successfully established based on the FliC protein obtained from prokaryotic expres-sion.关键词
肠炎沙门菌/FliC/原核表达/间接ELISAKey words
Salmonella Enteritidis/FliC/prokaryotic expression/indirect ELISA分类
畜牧业引用本文复制引用
赵东旭,蔡梦雷,苏士炫,刘逸洋,闫雪敏,张政钢,薛晓阳,闫金坤,崔国林..肠炎沙门菌fliC基因的原核表达及其间接ELISA方法的建立[J].中国兽医科学,2025,55(1):65-72,8.基金项目
河北省创新能力提升计划项目(235A6601D) （235A6601D）
中央引导地方科技发展资金项目(216Z6602G) （216Z6602G）
国家现代农业产业技术体系项目(CARS-40) （CARS-40）
