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Brucellosis is a widespread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating,febrile illness in humans.Quarantine,slaughter and vaccination are mainstay measures for the control of brucellosis.Since the current major serodiagnosis for brucellosis is based on smooth lipopolysaccharide,distinguishing vaccinated and naturally infected animals remains difficult.As the Brucella suis strain 2(S2)vaccine can protect vaccinated animals against heterologous Brucella spp.infections,we screened differential diagnostic biomarkers in S2 membrane proteins.The membrane protein extracts from S2 was subjected to two-dimensional electrophoresis,immunoblotting with three sera pools from healthy(non-infected/non-vaccinated),S2 vaccinated,or naturally infected sheep,and MALDI-TOF MS analysis of protein spots with differential immunoreactivity.Meanwhile,the S2 membrane protein extracts was immunoprecipitated with the above three pools of sera and Q Exactive MS analysis of the collected participates.From the above two approaches,ten proteins were identified as candidates for differential diagnosis,and two of the ten proteins were identified using both approaches.After expression,purification and immunoblotting and ELISA analysis,three out of ten proteins were demonstrated as potential differential diagnostic biomarkers: universal stress protein,glycoside hydrolase family 43 and hypothetical protein(31-kDa outer-membrane immunogenic protein).In conclusion,these findings provide new biomarkers to develop sensitive and specific immunoassays for the serodiagnosis of brucellosis,with emphasis on differentiation between vaccinated and naturally infected animals.