Sero-investigation On Brucellosis In Large-scale Dairy Farms And Establishment Of Indirect ELISA Detection Method

At present, China mainly brucellosis diagnostic methods for bacteria isolated and cultured and serological testing, but don’t meet the large rapid detection and testing requirements, and prone to false positives. In order to better diagnosis and prevention of brucellosis, we need to study new detection methods to meet the rapid and large detection.1. Since January 2013-2014 In December 2009, this laboratory plate agglutination test methods for inspection and cows collected serum samples were tested, preliminary screening of the popular dairy brucellosis situation. Total number of samples for the detection of cow serum 8160 copies, of which this is the number of positive serum samples 1768, accounting for 21.67% of the total number of samples to detect. Abortion cow serum samples detected 898 copies, of which 400 positive serum samples, accounting for 44.54% of the total number of samples to detect abortion; cow serum samples to detect large parts of 7262, in which the number of positive serum samples to 1368 copies, accounting for 18.84% of the total number of samples to detect large.2. Brucella outer membrane protein OMP16 have good immunogenicity, is a popular candidate for subunit vaccines. T4SS is intracellular parasite must Brucella virulence factors. In 2011, Marchesini MI reporting systems such as the use CyaA identification BPE123, BPE275, BPE005 and BPE043 by T4SS secreted effector proteins. They play an important role in regulating the transport process within Brucella cell, whether it has a good immunogenicity has not been proven. Therefore, this study selected OMP16 and BPE123 two genes, purified recombinant protein, try to establish indirect ELISA method.According to the GenBank Brucella OMP16 gene sequences BPE123 designed specific primers OMP16 and BPE123 gene fragments. Cloning plasmids OMP16 and BPE123 gene, while the double digestion and DNA sequencing, the positive plasmid was named pET-T3-OMP16 and pET-T3-BPE123. While cloning plasmid pET-T3-OMP16, pET-T3-BPE123 and pET-32a (+) expression plasmid digested. Recycling target gene fragment OMP16, BPE123 and pET-32a (+) linear fragments, connecting them with a ligase to construct the expression plasmid pET-32a-OMP16 and pET-32a-BPE123. Then the two into a host bacterium E. coli expression construct bacteria pET-32a-OMP16-BL21 and pET-32a-BPE123-BL21. IPTG induction of the expression strain induced expression and optimized conditions. SDS-PAGE and Western-blot analysis of protein expression was identified. The recombinant protein was purified and collected a lot with Ni-NTASpin Kit. The purified protein OMP16 and BPE123 as coating antigen, try to build iELISA detection method. Trying to determine the best package antigen test is the concentration and optimal coating condition; determining optimal dilution of secondary antibody to determine the yin and yang of the threshold, and a test to determine the sensitivity and test repeatability. By iELISA established method to detect serum samples from 245 cows, and with the rose bengal plate agglutination test as a control, it is determined in line with the rate of the method.Purified protein OMP16 and BPE123 successfully constructed iELISA detection method OMP16 as coating antigen. The detection method and rose bengal plate agglutination test compliance rate of 91.38%. BPE123 protein when trying to establish iELISA detection methods can not distinguish between yin and yang serum appear more problems, iELISA BPE123 protein detection methods failed to build.Conclusion:brucellosis epidemiology survey, brucellosis has occurred in many pastures across the country, and the incidence has a clear upward trend; factors in causing abortion, brucellosis is very cause miscarriage cows important factor. Indirect ELISA method was successfully constructed OMP16 can be used as candidate detection method cows brucellosis serological testing; iELISA BPE123 protein detection methods failed to build, but can in order to further explore the immunogenicity and in Brucella intracellular parasitic and Functional foundation.