肠炎沙门菌fliC基因的原核表达及其间接ELISA方法的建立OA北大核心

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.