| Brucellosis (Brucellosis) is a worldwide zoonosis serious disease,whitch has an important significant in veterinary public health. For the establishment of a bovine brucellosis fast and reliable serological detection methods, this paper has been studied a recombinant proteins as mixed antigen-based Enzyme linked immunosorbent assay,whitch was based on its three kinds of outer membrane protein prokaryotic expression, the details are as follows:According to GenBank,three pairs of specific primers to OMP19, OMP22, OMP28 were designed,respectively.Brucella strains A544 gene as a template, utilized PCR to amplify the gene fragment.The target gene was cloned into pCold-TF prokaryotic expression vector system, after verified correctly by PCR, enzyme digestion and DNA sequencing, the recombinant proteins were induced and expressed by the IPTG? analysed by SDS-PAGE and Western blot, then were purified by affinity chromatography.The results showed,534 bp,639 bp and 753 bp encoding abortus outer membrane protein OMP19, OMP22, OMP28 gene sequences was successfully amplified, and cloned into pCold-TF prokaryotic expression vector pCold-TF-OMP19, pCold-TF-OMP22 and pCold-TF-OMP28 for recombinant gene expression plasmid of three proteins. After being induced and expressed.three recombinant proteins'molecular weight were 69 kD,74 kD and 80 kD. all of whitch were soluble proteins, the result of Western blot showed whtich had good reactogenicity. Affinity chromatography was used to purify three recombinant proteins, then we obtained high-purity protein.The three recombinant proteins were at a concentration ratio. mixed in equal proportions as coating antigen,after optimizing the coating condition, closing conditions and the reaction temperature and time, we initially established an indirect ELISA method for antibody detection of B. abortus infection.and laboratory saved 300 clinical samples were detect application,the method was parallel compared with bengal plate agglutination test (RPAT) and similar Commercially available reagents (IDEXX), to verify sensitivity and specificity of the method. The results showed that the established ELISA method, the optimum concentration of antigen was combined by the concentraintion of (1:1:1) ?g/ml, the optimum blocking solution was 10% horse serum, optimal antibody dilution was 1:200. optimal HRP-Comjugate dilution was 1:20,000.The method showed good specificity, and not cross-react with Yersinia enterocolitica and E.coli diagnosis diagnostic serum.Bengal plate agglutination test (RBPT) standards to IDEXX kit as a reference to the establishment of detection methods for parallel detection control 300 clinical serum samples. Results of RBPT showed,62 samples were positive,238 samples were negative. Results of IDEXX test kit for the detection of 62 positive serum samples showed,60 samples were positive,2 samples were false negative, the sensitivity was 96.78%(60/62); the detection of 238 negative serum samples showed,236 samples were negative.2 samples were false-positive, specificity was 99.16%(236/238), total coincidence rate was 98.67% (96/300). Test results of the rOMPs-ELISA method for the detection of 62 positive serum samples showed,58 samples were positive,4 samples were false negative, sensitivity was 93.5%(58/62); the detection of 238 negative serum samples showed,230 were negative and 8 were false positives, the specificity was 96.64%(230/238), total coincidence rate was 96%(288/300).This research lays the fondation for application and further commercialization of Brucella antibody test indirect ELISA. |
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