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

作　　者：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 ZHAO Dongxu;CAI Menglei;SU Shixuan;LIU Yiyang;YAN Xuemin;ZHANG Zhenggang;XUE Xiaoyang;YAN Jinkun;CUI Guolin(School of Life Sciences and Food Engineering,Hebei University of Engineering,Handan 056038,China;Huayu A gricultural Science and Technology Co.,Ltd.,Handan 056000,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,河北邯郸056038 [2]华裕农业科技有限公司,河北邯郸056000

出　　处：《中国兽医科学》2025年第1期65-72,共8页Chinese Veterinary Science

基　　金：河北省创新能力提升计划项目(235A6601D);中央引导地方科技发展资金项目(216Z6602G);国家现代农业产业技术体系项目(CARS-40)。

摘　　要：建立一种基于肠炎沙门菌鞭毛蛋白FliC的间接ELISA方法。在大肠杆菌BL21(DE3)中表达肠炎沙门菌FliC截短蛋白,将其作为包被抗原建立间接ELISA方法;采用52份临床阴性鸡血清测定该方法的阴阳性阈值,采用沙门菌阳性鸡血清测定该方法的特异性、敏感性和重复性,采用135份临床血清样品测定该方法与BioChek公司禽沙门菌D群ELISA试剂盒检测结果的符合性和临床适用性。结果显示:截短表达的FliC蛋白在BL21(DE3)中呈可溶性表达,可与H:g抗体特异性结合,与H:i和H:m抗体不反应;基于截短表达的FliC蛋白的间接ELISA方法包被抗原最优质量浓度为4 ng/μL、待检血清最优稀释度为1∶40、HRP标记的第二抗体的最优稀释度为1∶2000;该方法的阴性阈值为0.82,阳性阈值为0.94,可检出80倍稀释的肠炎沙门菌阳性血清抗体,未检出鸡白痢沙门菌、鼠伤寒沙门菌、姆班达卡沙门菌、汤姆森沙门菌和肯塔基沙门菌阳性血清抗体,批间和批内重复检测变异系数分别为0.43%~5.13%和0.50%~2.68%,与禽沙门菌D群ELISA试剂盒的检测符合率为94.1%。本研究通过原核表达获得FliC截短蛋白,并基于该蛋白成功建立了一种检测肠炎沙门菌抗体的间接ELISA检测方法。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 Bio Chek 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∶2000.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 expression.

关 键 词：肠炎沙门菌 FLIC 原核表达 间接ELISA 

分 类 号：S852.612[农业科学—基础兽医学]