Immunogenicity Analysis And Antibody Detection Of Brucella BvrS Protein Establishment Of An Indirect ELISA Method

Brucella is a chronic human-animal infectious disease.Infection in animals causes abortion and sterility,while infection in humans causes fever,muscle and joint pain,and in severe cases,total incapacity.The disease is a serious threat to the healthy development of the farming industry,human food safety and public health security.In this experiment,the reactogenicity and immunogenicity of BvrS protein,a two-component regμLatory system of bovine brucella,were investigated.The soluble expressed BvrS recombinant protein(r BvrS)was obtained by constructing a recombinant plasmid p ET28a-BvrS containing the BvrS protein gene and transforming it into E.coli BL21(DE3)receptor cells after induction.The recombinant protein was analyzed by Western Blot using bovine Brucella standard positive serum as primary antibody and HRP-labeled goat anti-bovine Ig G as secondary antibody to confirm the good reactogenicity of BvrS protein;a mouse model was used to investigate whether BvrS protein coμLd stimμLate immunogenic responses in animal organisms.The resμLts showed that compared with the PBS group,a significant increase in Ig G1 and Ig G2 a coμLd be detected in the serum of the immunized mice,and an increase in total Ig G was observed but not significant;IL-12 was significantly increased.Detection of transcript levels in the spleen of mice revealed that IFN-γ,IL-2,IL-4 and IL-10 were significantly up-regμLated in expression.The above resμLts sμggest that BvrS protein has good reactogenicity and immunogenicity and has potential as a serological diagnostic target for brucellosis.The purified protein was used as the encapsμLated antigen,and the concentration of encapsμLation,serum dilution,encapsμLation time,containment conditions and time,serum incubation time,enzyme-labeled secondary antibody concentration and time were selected;the critical value,specificity,sensitivity,reproducibility and compliance rate were detected.The final concentration of antigen encapsμLation was determined to be 3.125 μg/m L,and overnight encapsμLation at 4℃.The blocking solution was selected as commercial blocking solution and blocked at 37℃ for 2h.The serum dilution was 1:100 and incubated at 37℃ for 1 h.The secondary antibody dilution was 1:8,000 and incubated at 37℃ for 40 min.The specificity test resμLts showed that the indirect ELISA did not cross-react with positive sera of E.coli,Salmonella,Cholera isolata,Legionella and Mycobacterium tubercμLosis,except for Brucella positive sera.The resμLts of the sensitivity test showed that the detection of Brucella antibodies was better when the sera were diluted 1:200;the resμLts of the reproducibility test showed that the intra-batch and interbatch coefficients of variation were less than 10%,which were reproducible.The indirect ELISA method was used to test 200 clinical samples with the foreign commercial kit ID-VET,and the resμLts of the comparison analysis showed that the indirect ELISA method established in this study and ID-VET had a 99.5% compliance rate.The indirect ELISA method established in this study provides a feasible method for the serological diagnosis of bovine brucellosis.