| Brucellosis is a zoonotic disease that is caused by Brucella spp.. It is severe affecting humans health and the development of animal husbandry. Data from WHO showed that the economic loss caused by Brucellosis is over three billions. Until now, the Malta Fever and chronic infection, abortion and sterility in ruminant can not be cured. Moreover, this pathogen has been used to develop biological weapons by terrorists. Therefore, Brucellosis is a severe threaten to the economy construction, public health and the national security to our courtry. One important characteristic is that once infected, it is hard to cure completely. Prevention is the most important means to control it. A problematic aspect of traditional serological detection is false-positive serological reactions (FPSRs). These FPSRs find their origins in the cross-reactivity observed between the S-LPS of Brucella species and the LPSs of some other Gram-negative bacterias. The common serological diagnostic methods of Brucellosis also can not distinguish from the vaccine or natural infection. The development of a tagged vaccine and the search for suitable diagnostic antigen has been the people's goals.BP26 is an important diagnostic antigen for Brucella. It is extensively used in Brucella diagnosis. It is a periplasmic protein, and not only play no roles in virulence, but also possess the good antigenity. These characteristics make it a good candidate antigen for gene tagging vaccine development. The deletion of this antigen can not only decrease the virulence, but also provide a means for differentiation.In the present study, the recombinant pET-30a(+)-bp26 and pGEX-6P-1-bp26 protein were expressed and purified by affinity chromatography. The recombinant pET-30a(+)-bp26 protein was used to immunize female BALB/c mice. Spleen cells harvested from BALB/c mice immunized with rBP26, were fused with SP2/0 myeloma cells using PEG as the fusion agent. The culture supernatants from hybridoma cells were tested for reactive MAbs by I-ELISA using the recombinant pGEX-6P-1-bp26 protein. After four sub-clones, five hybridomas were established and named 3AG5,3AG6,3CE10,4EA2 and 4EF3. All of them were IgG1 in nature. These MAbs did not show any cross-reaction with the whole cell lysate of Escherichia coli O157, Y.enterocolitica O:9, Salmonella and so on. When the five MAbs were tested by Western blotting, it was not recognized by the whole cell lysate of B.melitensis M5-90-â–³26 but a specific band was seen when the whole cell lysate of B.melitensis M5-90 was used. These data suggested that the five MAbs may have the potential function in the detection of Brucella species with high specificity.In this study, it was found that the diagnostic results of detecting B.abortus were fairly satisfactory by preliminary application of these monoclonal antibodies in competitive ELISA. It was expected to be applied to the clinical detection of Brucella. According to this study, a new serological diagnostic method of Brucellosis can be established and distinguish from natural infection or vaccination. It also can provide a theoretical basis for the application and diagnosis of the two tagged strains deleted the bp26 gene which were constructed in this study group.
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