Secretory Expression Of VirB12 From Brucella Bovis And Establishment Of Indirect ELISA

Brucellosis is a zoonotic infectious disease caused by brucella,the disease occurs all over the world,and it is very popular in our country.Abortion is the main symptom of the disease in livestock,symptoms such as wavy fever,arthritis and endocarditis occur in humans.Several animal brucella vaccines exist at present,such as Brucella vaccine attenuated 2,Brucella abortus A19 attenuated vaccine and so on,they are also widely used in China,it has played an important role in the prevention of brucellosis in cattle.However,they generally have some shortcomings,such as low protection rate for animals?high virulence to immune workers?lack of differential diagnostic markers and inability to distinguish immune animals from wild-infected animals.In recent years,researchers have done a lot of research,development of multiple gene deletion vaccines on the basis of original vaccines.At present,the A19-?VirB12 gene deletion vaccine based on A19 vaccine has entered the approval stage of new veterinary drugs,it is expected to be approved and popularized in the near future,this will lead to a huge demand for detection of VirB12 antibodies in vaccinated livestock and detection of wild virus infections.However,no corresponding detection method has been applied yet.The purpose of this study is to establish a method for identification and detection based on VirB12antigen.In order to obtain a highly purified and easily purified VirB12 protein antigen of Brucella,primers were designed and amplified according to the gene sequence of VirB12 in Gen Bank.The amplified product was linked to p BIC plasmid and transformed into Bacillus pumilus.The secretory expression strain of VirB12 protein was obtained.After induction,they were identified by PAGE and Western-blotting.The results showed that the secretory expression strain of VirB12 was successfully constructed and expressed in the culture supernatant after induction.The protein content was up to 80%.It could react with the standard positive serum of Brucella and the monoclonal antibody of 6×His,but did not react with the immune serum of A19-?VirB12 strain.In order to obtain high purity VirB12 protein of Brucella,VirB12 secreted and expressed protein was purified by affinity purification and ion exchange,the VirB12 recombinant protein with high purity was coated with ELISA plate.After optimizing the conditions,an ELISA method for detecting brucella antibody was established.The results showed that the two-step purification of VirB12 recombinant protein was very effective,and the purity of VirB12 recombinant protein could be more than 98%.In the established ELISA,the optimum coating concentration of the purified recombinant protein was 1mg·m L^-1,the optimum sealing solution is PBST containing 5%skimmed milk powder,the optimum dilution concentration of serum was 1:25,and the optimum dilution concentration of enzyme-labeled antibody was 1:1500.The results of 20 positive sera and 12negative sera showed that the overall coincidence rate with SUANOVIR Brucella-Ab C-ELISA kit was 90.6%.In order to analyze the specific antigenic epitopes of VirB12 protein and to make full use of the advantages of high purity and easy synthesis of specific polypeptides,the study of VirB12 polypeptide ELISA was carried out.Ten polypeptides were designed and synthesized according to the sequence of VirB12 gene.The synthesized 10 polypeptides were used as antigens,screened preliminarily by dot-blotting test and verified by ELISA.Finally two polypeptides P07 and P08 with reactivity were identified.P07 and P08 were labeled with biotin.P07B and P08B were labeled with biotin as antigens respectively.ELISA experiments were carried out with the enzyme label plate coated with avidin.It was found that the reactivity of P07B polypeptide was better than that of P08B polypeptide.Optimized conditions for using P07B polypeptide as antigen,the ELISA method for detecting Brucella antibody was preliminarily established.In the established ELISA,the best concentration of avidin is10mg·m L^-1.The optimal concentration of P07B peptide is 1mg·m L^-1.The best blocking solution is PBST solution containing 10%porcine serum.The optimal dilution concentration of 1:100 and the optimal concentration of enzyme antibody is 1:3000.The results of 19positive sera and 13 negative sera showed that the overall coincidence rate with SUANOVIR Brucella-Ab C-ELISA kit was 87.5%.The results of the above experiments show that,the ELISA method based on the secretion,expression and purification of VirB12 and P07B polypeptides as antigens can detect brucella infection antibodies.Detection of wild virus infection from livestock immunized with A19-?VirB12,it is hopeful to develop a detection kit for detection and purification of brucellosis in aquaculture farms.