Detection Of Brucella Antibody Based On WZT Recombinant Protein

Brucella is an important zoonotic disease that is widespread worldwide and can cause symptoms such as abortion,arthritis,and orchitis.The disease is particularly important because it can infect human.After infection,people will show symptoms such as fever,limb weakness,joint pain and so on.The pathogenesis and immune mechanism of brucellosis are still not very clear and it is difficult to treat by antibiotics.At present,the scheme for Brucella is based on prevention,so the precise detection of Brucella infection is particularly important for controling this disease.The first part of the study was to screen for a good target antigen.Lipopolysaccharide(LPS)is the main virulence factor of Brucella,and the gene in lipopolysaccharide is highly conserved,so a suitable gene is selected from lipopolysaccharide for use as a diagnostic antigen.The pre-screened gene was cloned into the pET-30 a vector and then transferred into the E.coil(BL21)expression vector,and the recombinant protein was obtained by inducing expression of IPTG at 25?.The available proteins were screened by Western blot based on their reactivity.The recombinant WZT protein was selected as suitable antigen to detect the corresponding antibody.The second part of the study was the establishment of an indirect ELISA method for Brucella.The selected antigens were gradually optimized to establish an indirect ELISA method for Brucella.The optimal coating amount of antigen was 15 ?g/mL,the optimal dilutions of serum was 1:80,the optimal dilution of enzyme-labeled secondary antibody was 1:5000.It was found that the coincidence rate of the ELISA method established in this study and the tiger red agglutination test was 96%.This method was also used to detect Yersinia enteric-positive sera and E.coli-positive sera,and proved to have good specificity.The third part of the study was to develop a colloidal gold immunochromatographic test strip for detection of Brucella infection.The colloidal gold particles(28nm)of suitable size were prepared by trisodium citrate reduction method,and the G protein was labeled with Streptococcus mutans G protein antibody as the quality control line(C line)after purification to the optimum pH value.The recombinant protein was lined on a nitrocellulose membrane as a detection line(T line).This method was used to detect Yersinia enteric-positive sera and E.coli-positive sera,and was found to have good specificity.