| Brucellosis is a zoonotic disease caused by members of the genus Brucella, and the main syptoms of the infected animals are abortion and dysgenesia. Recently, Brucellosis is prevalent in some fields of China, which brings a great threat to the development of livestock industry and human health. To prevent and eradicate the disease effectively, it’s very important to diagnose it rapidly and accurately. Rose bengal plate agglutination test (RBT), serum tube agglutination test (SAT) and complement fixation test (CFT) are still the main methods for diagnosing brucellosis in China. Thus, it is necessary to develop ELISA methods to detect serum antibody against Brucella, which may complete a high-throughput and precise detection. In our study,5specific proteins of Brucella were expressed by prokaryotic system, and their feasibilities for diagnosis were estimated. Finally, the recombinant protein BP26was selected, and an indirect ELISA for diagnosing brucellosis was established.1. The expression of recombinant proteins BP26, OMP16, CP39, SP41and eryCAccording to the sequence of BP26,OMP16, CP39, SP41and eryC gene of B. melitensis M5, five pairs of primers were designed. The target fragments with expected sizes of868bp,556bp,732bp,1329bp and985bp were amplified by PCR, and were cloned into pMD18-T vector respectively. Then, they were subcloned into the downstream of T7prometer of pET-28a(+) vector. The recombinant plasmids were transformed into E.coli BL21, and the recombinant proteins were induced by IPTG. Various expression conditions were optimized systematically, and then the expression products were analyzed by SDS-PAGE. The results showed that the molecular weights of the expression products were about32kDa,21kDa,30kDa,51kDa and38kDa, respectively. The best expression conditions of recombinant proteins were as following:The recombinat E.coli BL21was induced with1mM IPTG at37℃for5 hours.The expression products were purified from bacterial inclusion bodies by His·Bind affinity chromatography, and the concentrations of purified BP26,OMP16, CP39, SP41and eryC were1.0mg/mL,2.4mg/mL,3.5mg/mL,1.3mg/mL and1.7mg/mL, respectively. The result of western-blotting showed that all the recombinant proteins could react with the cattle antiserum against Brucella.2. The establishment of an indirect ELISA for the detection of antibodies against BrucellaFive purified recombinant proteins BP26, OMP16, CP39, SP41and eryC were used to coat microtiter plates with concentration of0.5μg/mL,4μg/mL,3μg/mL,4μg/mL and2μg/mL, respectively.Forty-eight cattle serum samples were tested. The sensitivities of the methods were90.9%,90.1%,97%,90.1%and90.1%, respectively, when compared with. Pourquier(?) ELISA Kit of IDEXX, and their specificity were80%,80%,73.3%,73.3%and53.3%, respectively. Then twenty positive sera and eight negative serum, which were detected by RBT、SAT and Pourquier(?) ELISA Kit were further detected by ELISA coated with the five proteins. By analysis of the data, the P/N value of recombinant protein BP26was the highest one among the five proteins. Therefore, BP26was selected as a coating antigen.The optimal coating concentration of antigen was500ng/mL and the dilution of serum was1:100, which were determined by checkerboard titration. The cutoff value was determined as0.2×ODP+0.8×ODN. When cattle sera against Mycobacterium tuberculosis, Escherichia coli, Foot-and-mouth disease virus and bovine viral diarrhea virus were tested, the results of the iELISA were all negative. The results of reproducibility showed the coefficient of variation (CV) was less than10%. Finally, a total of174clinical cattle sera were detected. The positive rates detected by the established iELISA and Pourquier(?) ELISA Kit were68.97%and70.11%, respectively. Compared to Pourquier(?) ELISA Kit, the sensitivity, specificity and coincidence rate of the iELISA were94.2%(113/120),83.3%(45/54) and90.8%(158/174), respectively.All these results indicated the iELISA based on recombinant periplasm protein BP26was sensitive and specific, and reproductive. The iELISA could be used to detect the antibody of cattle sera against Brucella. |
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