Establishment Of An Indirect ELISA Method For Detection Of Bovine Serum Antibodies Using RGapC_1-150 Of Streptococcus Agalactiae Or Its Tandem Epitope Peptide As Antigen

Bovine mastitis is mainly caused by microbial infection,which seriously impedes the development of dairy industry in the world.The main microorganisms causing cow mastitis include streptococcus,staphylococcus,enterobacterium and so on.Streptococcus is one kind of the most common pathogens causing bovine mastitis.Due to the long-term use of antibiotics to prevent and treat bovine mastitis,the drug-resistant streptococcus strain is increasing rapidly,and leading to poor drug treatment for the strain infection.Therefore,great efforts have been made using vaccine to prevent the bovine mastitis caused by streptococcus.A fusion protein GIT,a vaccine candidate for bovine mastitis,constructed using the recombinant streptococcus Gap C_1-150as a part of GIT is under clinical trial now.However,there is no effective detection method for the antibody of cow immunized with the vaccine candidate,and the humoral immune response to GIT can not be effectively evaluated.Therefore,we use the recombinant Gap C_1-150 protein of Streptococcus agalactiae or the peptide composed of two B cell epitopes in tandem from Gap C_1-150 as antigen to establish indirect ELISA method for detection of antibody in the immunized bovine serum.The Gap C_1-150 gene fragment from Streptococcus agalactiae HLJ57 strain was amplified by PCR,inserted into p ET-32a vector and transformed into competent cell BL21?DE3?.The expressed Gap C_1-150 protein was purified,and emulsified with equal volume of Freund's adjuvant.Healthy Holstein cattle of 6-18 month old were intramuscularly inoculated with the emulsified immunogen,and the cattle blood sample was collected on a day from 10-14 days after boosting immunization for serum preparation.The Gap C_1-150 protein expressed with p GEX-6p-1 vector was used as detection antigen to establish an indirect ELISA method.The results showed that the optimal concentration of coating antigen was 2.5?g/m L,and the optimal serum dilution was1/1000.The coating solution was PBS?p H 7.2?,and the coating time was 37?45 min.The blocking solution was 1%BSA+0.05%Tween 20,and the blocking time was 37?1.5 h.The optimal serum incubation time was 37?30 min.The cut-off value was determined as 0.487according to analysis of OD₄₅₀ value from negative sera and positive sera.The serum from bovines immunized with Gap C_1-150 or GIT were positive,and the serum from bovine immunized with Trap,Isd B,p ET32a tag protein,Staphylococcus aureus wood46,Streptococcus agalactiae HLJ57,or serum of cattle suffered from E.coli diarrhea,paratuberculosis,or brucellosis were negative by the ELISA detection.When the dilution of positive serum was 1/16000,the P/N value was still greater than 2.1.The intra assay coefficient of variation was 3.6%-9.6%,and the inter assay coefficient of variation was 1.59%-9.81%.We detected 85 sera from clinical by the ELISA method,and the serum positive rate was 10.59%.In order to establish a more specific and sensitive ELISA method for the detection,two B cell epitopes from Gap C_1-150 were linked with the AAY amino acid residues to form a novel peptide,³⁰TRINDLT³⁶AAY⁴⁶DTTQGRFDGT⁵⁵?named TT-20?,as the antigen to establish an ELISA method.The results showed that the optimal concentration of the coating antigen was 15?g/m L,the serum dilution was 1/800,and the cut-off value was determined as 0.36 according to analysis of OD₄₅₀ value from negative sera and positive sera.When the dilution of positive serum was 1/6400,the P/N value was still more than 2.1.There was no cross reaction between the coated peptide TT-20 and the serum of bovine immunized with each of the antigens except Gap C_1-150 or GIT,or the serum of the cattle suffered from E.coli diarrhea,paratuberculosis,or brucellosis.The intra assay coefficient of variation was 1.4%-3.6%.The positive rate of 85 sera from clinical was 17.65%by the ELISA detection.The results above demonstrated that the established indirect ELISA with Gap C_1-150 as antigen is specific and more sensitive than that with peptide TT-20 as antigen,and can be used for the detection of serum antibody from cattle immunized with streptococcal Gap C_1-150 or GIT.