| Brucellosis is a zoonotic infectious disease caused by the gram-negative,facultative intracellular parasite Brucella,which is widely prevalent all over the world.It seriously threatens human health and causes huge losses to animal husbandry.Brucella can infect humans,cattle,sheep,pigs,deer,dogs and a variety of wild animals.Humans are mainly infected through sick animals and their products.In 2016,the Ministry of Agriculture and National Health and Family Planning Commission issued the"National Brucellosis Prevention and Control Plan(2016-2020)"In 2022,the"Five-Year Action Plan for the Prevention and Control of Brucellosis in Animals(2022-2026)"was issued.Strengthening animal brucellosis monitoring and early detection and elimination of positive individuals are one of the important measures for brucellosis prevention and control.In order to establish detection methods such as competitive ELISA,this study selected Brucella outer membrane protein 16 with high conservation and good immunogenicity.The structure,physicochemical properties or B cell linear epitope of OMP16 were analyzed by bioinformatics software;Construction of prokaryotic expression vector of Brucella OMP16 by homologous recombination and its expression and purification;eight OMP16-specific hybridoma cell lines were obtained by the mouse hybridoma technology and purified OMP16 monoclonal antibody was obtained;a competitive ELISA detection method for Brucella OMP16 antibody was initially established.The test results are as follows:(1)The nucleic acid or amino acid sequence of B.suis.S2 strain OMP16 was obtained from the NCBI website.The Prot Param software predicted that OMP16 was composed of 168amino acid,the relative molecular mass was 18 ku,the protein Isoelectric Point PI was 9.92,the half-life in E.coli was more than 10 h,the coefficient is 43.86,which belongs to unstable protein,and the fat coefficient is 78.57,which belongs to lipoprotein and has good thermal stability;TMHMM and Signal P predict that OMP16 has a signal peptide sequence at 1～24amino acid,and there is a transmembrane region at 6～28 amino acid;IEDB predicts OMP16has seven B cell linear epitopes,located at 6～8,27～51,62～72,87～88,100～110,128～130,136～155 amino acid.Cloning of the uomp16 sequence without the signal peptide and transmembrane region,the p GEX-4T-1-uomp16 prokaryotic expression vector was constructed by homologous recombination method,transferred into E.coli BL21,induce 8h with 1mmol/L IPTG at 22℃,successfully expressed,and obtain purified r GST-u OMP16 by GST resin affinity chromatography;Amplify the prokaryotic expression vector p ET32a-omp16,transfer it into Escherichia coli BL21,induce 8h with 1mmol/L IPTG at 22℃,successfully express,and obtain purified r His-OMP16 by NTA resin affinity chromatography;Western-Blot results showed that r His-OMP16 and r GST-u OMP16 had good reactogenicity.(2)After emulsification of rHis-OMP16 with Freund’s complete adjuvant or Freund’s incomplete adjuvant,the mice were immunized,and the OMP16 antibody level in the serum of the mice was detected by i ELISA to reach 1×10~6～10~7.rGST-uOMP16 was used as the coating antigen,and the mouse immmune/negative serum was used as the primary antibody,and the optimal antigen coating concentration of i ELISA was determined to be 0.5μg/ml,and the optimal dilution ratio of enzyme-labeled secondary antibody was 1∶5000.The i ELISA method for OMP16 antibody screening was established.Mouse peritoneal macrophages were used as feeder cells,immunized mouse spleen cells were fused with SP2/0 cells under the action of 50%PEG 4000,and eight OMP16-specific hybridoma cell lines were obtained through hybridoma screening,which were named B7,D1,D2,D3,E6,F5,H4 and H11.Among them,B7,D1,D3 or F5 is Ig M-κtype,D2,H4 or H11is Ig G1-κtype,E6 is Ig G2b-κtype,and the hybridoma cells have normal karyotype and high specificity of monoclonal antibody.(3)Optimize the antigen coating concentration,antibody dilution ratio,serum dilution ratio,enzyme-labeled secondary antibody dilution ratio or optimal blocking conditions,detect28 clinical negative sera of goat,determine the critical value,and preliminarily establish Brucella OMP16 antibody competition ELISA:0.625μg/m L r GST-OMP16 100μL/well,coated overnight at 4℃;TBST Buffer 200μL/well,washed 3 times;3%Gelatin blocking solution 100μL/well,blocked at 37℃ for 2 h;TBST Buffer 200μl/well,washed 3 times;antibody 1∶200 dilution,serum 1∶10 dilution,100μL/well after mixing,incubated at 37℃ for 1 h;TBST Buffer 200μL/well,washed 3 times;enzyme-labeled secondary antibody 1∶15000 dilution,100μL/well,incubate at 37℃for 1 h;TBST Buffer 200μL/well,wash 3times;TMB chromogenic solution 100μL/well,incubate at 37℃for 30 min;2M sulfuric acid stop solution 50μL/well,stop the reaction,when PI%≥39.2%,it was judged as positive;when PI%<39.2%,it was judged as negative,32.2%<PI%<39.2%,judged as suspicious.60goat clinical serum samples were tested,and the compliance rate was 95%compared with the third-party test results,and the results were well consistent.This study expressed Brucella OMP16,screened OMP16-specific hybridoma cell lines,and established an OMP16 antibody competition ELISA method,which is expected to provide a detection method with high specificity and sensitivity for clinical prevention and control of Brucella. |
