Preparation Of Brucella Abotus Lipopolysaccharide Monoclonal Antibody And Functional Study Of Acid Shock Protein 24

Bovine brucellosis is a worldwide zoonotic disease,caused by Brucella abortus which is a Gram-negative,facultative intracellular bacterium.Brucella has no classical virulence factors,such as plasmid,capsules and exotoxins,whose virulence relies on its ability to invade into and survive within host cells.Investigation of Brucella pathogenesis is important for prevention and control of brucellosis,and at present,differential diagnosis and development of novel effective vaccines are two big issues which need to be solved in control of brucellosis.The diagnosis of brucellosis in humans as well as in animals is usually based on the detection of specific antibodies against lipopolysaccharide(LPS)of Brucella.In this study,we had acquired a positive hybridoma cell by the fusion of spleen cells and sp2/0 cells,then screened monoclonal antibody against Brucella abortus LPS by ELISA,named 51C.Cross reactivity experiments showed 51C monoclonal antibody can react with LPS of Brucella abortus S2308 and Brucella suis 1330,but it can not react with LPS of B.melitensis 16M,E.coli 0157 and Salmonella typhimurium SL1344,and it reacted slightly with LPS of Y.enterocolitica O:9.Thus,we speculated that 51C monoclonal antibody in this study may react with the A epitope of LPS.The monoclonal antibody we prepared was beneficial for establishing a specific detection method of brucellosis in further study.Asp24 is another important virulence-related protein in Brucella,which can be induced expression at acidic condition.As a candidate vaccine strain,Δasp24 mutant has more potential application.In our previous study,we found that asp24 enhanced expression when O-antigen accumulates intracellularly,which was in favor for Brucella intracellular survival.However,the function of Asp24 is unclear.In this study,we had constructed the asp24 mutant by bacterial directional homologous recombination.Growth curve showed that asp24 deletion in Brucella didn’t affect the bacterial growth.Intracellular survival assay demonstrated that asp24 was not associated with Brucella intracellular survival.In mice infection model,compared to the wild-type strain,the asp24 mutant was attenuated significantly at the late infection stage,but it didn’t show any difference in virulence at the early infection stage.In further study,we analyzed the condition of induced expression and the function of Asp24 protein.The results showed that Asp24 is down-regulated under the condition of heat stress.Promoter activity analysis showed the 89-bp fragment in front of open reading frame(ORF)is essential for Asp24 expression,and the 126-bp fragment in front of ORF is essential for Asp24 expression under acidic condition.Bioinformatics analysis found that Asp24 protein has three typical EF-hand domains which can bind calcium irons.To confirm this,we had expressed and purified Asp24 protein and three EF-hand domains mutant protein(Asp24-EF123)in vitro.SDS-PAGE analysis confirmed Asp24 can bind calcium ions.Asp24 protein when binding to calcium ions showed high electrophoretic mobility,however,the electrophoretic mobility of Asp24-EF 123 protein didn’t show any difference when Asp24-EF 123 protein binding calcium ions or not.Besides,we found that the expression of Asp24 was down-regulated in Brucella when exposed to the condition of calcium ions deprivation,and Asp24 was up-regulated expression when Brucella exposed to high concentration of calcium ions.It is suggested that Brucella may maintain homeostasis of intracellular calcium ions by regulating Asp24 expression.In conclusion,we prepared a monoclonal antibody against B.abortus LPS successfully,and proved that Asp24 plays a crucial role in establishing chronic infection in mice model,we further confirmed that Asp24 can bind calcium ions.This study prepared some materials for further establishing a specific diagnostic method of Brucellosis and provided novel insights into Brucella pathogenesis.