布鲁氏菌BP26蛋白原核表达及双抗原夹心ELISA方法的建立OA北大核心

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.