The Construction Of Brucella Protein Library And The Study Of The Identification Antigen Of Infection And Immunity Differential Diagnosis

Brucellosis is one of the zoonotic diseases, which harms seriously human health and the development of animal husbandry, caused by Brucella bacteria. The source of infection are livestock such as cattle, sheep and so on; Brucella propagates mainly through the ways such as the digestive tract, respiratory tract and skin mucous membrane, etc. Human and livestock are both susceptible.The golden standard in the diagnosis of brucellosis is the isolation of Brucella bacteria. However, culture sampling sensitivity is low, and Brucella is difficult to isolate. There is also a certain biological safety issues, so it is not suitable for rapid diagnosis and screening of a large area. Now the domestic clinical diagnosis of brucellosis combines rose-bengal plate agglutination test(RBPT) and standard serum agglutination test(SAT) together, which are both serological detection methods. But SAT is easy to cross-react with other bacteria, and time-consuming. Some researches found that enzyme-linked immunosorbent assay(ELISA) is more sensitive than SAT and complement fixation test(CFT). ELISA kits coated by smooth lipopolysaccharide of Brucella is developed to diagnose Brucella for cattle.But it is also easy to cross-react with other bacteria. So this study try to choose the monoclonal protein of Brucella which has good antigenicity to be as the antigen of indirect ELISA(iELISA), so as to built the method of iELISA coated by monoclonal protein, and provide a solid foundation for the further study on the rapid diagnostic of Brucellosis kit.Moreover, the mainly Brucella abortus is the naturally infected species for cattle. But the most universal cattle vaccines are M5and S2, which are brucella melitensis and brucella suis attenuated strain, respectively. The protein of Omp31is existed in all brucella species except Brucella abortus. So Brucella abortus infected serum can be distinguished from others by detecting the antibody of Omp31, and then differentiate between vaccination and natural infection.Now the protein researches of Brucella are various and the information is messy, so the protein library would be built at the beginning of this study, summarized the research of main Brucella protein, and the Brucella protein are provided into four parts:the outer membrane protein(OMP), Type â…£ secretion system(T4SS) protein, cytoplasmic binding protein and enzymes and others. And the functions of proteins are related to antigenicity and immunogenicity, the value for diagnosis, nosogenesis and so on. Then select the protein virB8which was rarely researched and is important for T4SS to express and purify. The method of iELISA was built to detect Brucellosis using the protein bp26, omp31(conserved in the laboratory) and virB8as antigens. The best concentration of antigen virB8, bp26and omp31were respectively12.90ug/ml,2.60ug/ml,1.24ug/ml; the optimal dilution of the serum of the three antigen were all1:100, and the optimal dilution of secondary antibody were all1:20000.70samples serum of heathy cattle from Daxing were tested as the condition above to confirm the cutoff value, and the result of virB8, bp26and omp31are respectively0.199,0.220,0.242.100samples serum from Henan first tested by RBPT and SAT, the result of56samples were positive. iELISA coated with virB8detected that14samples were positive, the sensitivity was only17.9%, the specificity was90.9%, so virB8had no practical value in the detection of brucellosis. iELISA coated with bp26detected that45samples were positive, the sensitivity was75%, the specificity was93.2%, consistency rate was83%, Kappa value was0.664, that showed these two methods have a high degree of consistency. The difference of these two detective methods was statistically significant by paired chi-square test, and SAT was better than iELISA coated with bp26. The result of iELISA coated with omp31was that all of100samples were negative including the56samples which tested positive by SAT. This inferred that all of the positive samples by SAT is caused by natural infection. Although iELISA coated with bp26had certain positive significance in the diagnosis of brucellosis, the sensitivity is not satisfied. The next step we will continue to screen the iconic antigen of Brucella, to explore the iELISA method of variety of antigen combined in the diagnosis of brucellosis.This study establishes the foundation for the systematic, comprehensive and authoritative Brucella protein library; provides a reference for screening monoclonal proteins to detect brucellosis by iELISA, provides a solid foundation for the further study on variety of monoclonal proteins combined to diagnose brucellosis; the result of this study provides a practical method to distinguish the cattle which is vaccinated or natural infection, and it has contributed to the development of animal husbandry and economic.