Screening Of Immunogenic Proteins In Brucella Abortus And Preliminary Study On Colloidal Gold Strip

Brucellosis is an important zoonosis characterized by abortion,sterility in ruminants and chronic infection and undulant fever in humans.It is widely distributed all over the world and causes threat to human and development of animal husbandry.At present,accurate diagnosis is the key strategy to control the disease.The sensitivity and specificity of traditional diagnostic methods are not ideal,and the experimental conditions are high.The common serological diagnosis methods lead to false-positive result.Therefore,it is of great significance to develop a sensitive,convenient and specific detection method for the diagnosis of brucellosis.In this study,we have completed three parts of research.1)Screening the previously reported main immunogenic proteins in Brucella.Firstly,the mouse infection model was established by intramuscular injection of Brucella abortus strain 2308 in BALB/c mice.The antibody production pattern of the OMP16,BP26,BLS,BCSP31,VirB12,SodC and GroEL proteins was evaluated at different infection stages of mouse model,and their application in the diagnosis of brucellosis was discussed.In further studies,the diagnostic effect of the seven proteins on 44 clinical bovine sera was evaluated using western blotting.2)Brucella novel immunogenic proteins were screened.Antibody production roles were assessed using western blotting in mice by infected with Brucella abortus at different infection stages.Immunogenic protein regions in Brucella were confirmed reacted with clinical bovine and caprine Brucella positive-sera by western blotting.Total Brucella proteins were separated by SDS-PAGE,and the immunogenic protein bands were incised from protein gel according to the results of western blotting.Subsequently,immunogenic protein SSB was successfully expressed and purified in E.coli strain.3)A colloidal gold strip was prepared in the study.Based on diagnostic results of BP26-ELISA,BLS-ELISA and LPS-ELISA,Brucella LPS was shown as optimal antigen,and then the LPS was extracted and purified,the colloidal goldparticles were prepared using sodium citrate reduction method,the colloidal gold conditions were optimized,such as blocking buffer,re-suspension buffer and types of NC membranes.Finally,SPG-linked colloidal gold particles were used,LPS as testing lines,sheep anti-mouse IgG as quality control line,a rapid colloidal gold strip was constructed in diagnosis of brucellosis.The results showed that 1)BP26 and BLS were the two best immunogenicity proteins among the 7 main immunogenic proteins.BP26 and BLS reacted with 30 Brucella-positive sera,but also produced false positive reaction with 14 Brucella-negative sera.In indirect ELISA,when BP26-ELISA was compared with LPS-ELISA,the coincidence rate of brucellosis positive serum was 92.68%,while that of negative serum was only 52.94%.however,it is difficult to distinguish Brucella-positive sera from negative sera by BLS-ELISA.At the same time,the use of different truncated BP26 protein fragments as antigens could not exclude the false positive reaction with Brucella-negative sera.2)Based on western blotting of Brucella reacted with infected mice sera,bovine and caprine sera,it was found that immunogenic proteins were mainly in two molecular weight regions of Brucella proteins,40-70 kDa and 7-20 kDa.Six Brucella immunogenic proteins were identified.Western blotting showed that SSB protein had good reactivity with Brucella-positive sera,and the coincidence rate with rose-bengal plate agglutination test(RBPT)was about 82%.3)a colloidal gold strip was prepared using purified LPS as antigen,and the condition of the optimum pH,protein labelled amount,NC membrane,stabilizing agents and re-suspension buffer were optimized,finally,a preliminary colloidal gold strip was constructed with successfully detection of bovine Brucella standard negative and positive serum.In summary,although BP26 and BLS are good immunogenic proteins of Brucella,BP26 and BLS as antigens in diagnosis of brucellosis may lead to false-positive reaction and misdiagnosis.The SSB protein had good reaction with bovine Brucella-positive sera,and the coincidence rate with RBPT was about 82%.This study laid a foundation for the development of diagnostic reagents for brucellosis.A colloidal gold strip was developed for the detection of brucellosis,and the diagnostic results were obtained within 5 minutes.