The Study Of Brucella Immonumagnetic Beads And Immunofluorescence Detection Methods

Brucellosis is one of important infectious zoonosis, which is caused by Brucella.It belongs to the second class infectious disease. Since 1980 s, with the development of animal husbandry, Brucellosisis is becoming increasingly prevalent and detrimental to humans. In recent years, Brucellosis mainly break out on small scales instead of large scales, which bring great difficulties to the control and prevention of this disease.Brucellosis not only seriously threatens human health, but also impacts the dairy, meat and animal husbandry industry, bringing huge challenges to animal food safty and public health.It takes complex procedure and much time to detect Brucella in animal-ori gin food. Immunomagnetic separation technology is a new technique which co mbines the sepecificity of immunological reaction with specific mangnetic respo nsiveness of magnetic beads. It can effectively collect and concentrate small a mount of pathogenic microorganisms in massive samples and separate target ba cteria with high separation rate and specificity, simple operation as well as exp ensive equipment-independence. In this study, we intend to establish Brucella I mmunomagnetic separation technology and explore the application in the detecti on of production of animal origin.bcsp31( Brucella cell surfacial protein 31) gene was amplified by PCR based on genomic DNA of B. Melitensis srtain M5 and cloned into vector pJET1.2, then was subcloned into expression vector pET28 a. The recombinant plasmid pET28a-bcsp31 was identified by digestion with NdeI/EcoRI and sequence respectively, and then was transformed into E.coli BL21(DE3) host cells and induced by IPTG, the recombinant protein BCSP31 was studied by SDS-PAGE and purified through Ni-NTA affinity chromatography. The immune response of purified BCSP31 was evaluated by ELISA.The results showed that the recombinant protein BCSP31 was expressed in soluble form. The rate of the expressed protein in the soluble bacterial protein was about46.3%. The serum antibody titer of BCSP31 immunized mice was 1:16 000 and can reaction with Brucella abortus M5.Recombinant protein BCSP31 and B. melitensis srtain M5 were used to immune rabbits respectively. Then polyclonal antibody was acquired and purified from the serum of immuned rabbits and was coated to magnetic beads. The optimal conditionsto prepare IMBs were explored. The results showed that activation for 45 min, 500 μL of antibody, coupling for 14 h, 20 μL of IMBs, response for 45 min, and pH 7.4 of washing solution were best to prepare IMBs. 9 concentration gradient ranging from10~109 CFU/mL of B. melitensis srtain M5 bacterium were captured using coated IMBs with recombinant protein BCSP31 antibodies and B. melitensis srtain M5 antibodies respectively. Then the mixture were coated on TSB plates and cultivated in incubator for 5 days. Suspected colonies were picked and identified by PCR, the detection limit of IMBs coated with BCSP31 antibodies was 104CFU/mL, while the detection limit of the IMBs coated with the M5 antibodies was 10CFU/mL, indicating the IMBs coated with the M5 antibodies worked better. IMBs can also detect B.abortus strain 45/20 and B. suis S2. Milk and meat sample artificially contaminated with M5 were also detected by IMBs and the results showed IMBs can detect as few as 103CFU/mL of B. melitensis. The direct PCR assay without IMBs was 104CFU/mL.The results indicates that IMBs capture combined with prolification on TSB plate could not only increase the detection sensitivity compared with PCR assay, but also isolate Brucella from contaminated samples directly.Besides, this study has established indirect immunofluorescence assay for the detection of Brucella based on the polyclonal antibody, and the dilution ratio of primery and secondary antibody were 1:40 and a minmum of 105 CFU/mL Brucella could be detected through fluorescence microscope. This assay could detect Brucella in animal viscera in as few as 2 hours.Taken together, recombinant protein BCSP31 were expressed and it proved to have favorable immunogenicity though ELISA. IMBs detection method was established using magnetic beads coated with antibody and it could not only i mprove detection sensitivity but also isolate Brucella in samples. Indirect immu nofluorescence assay can quickly detect Brucella, which saves much time. If w e conbined these two methods, it would be more effective in detecting the pre sence of Brucella from samples, providing a more convenient method for the market quarantine, and has a great significance for the magnitude public health incident.