Function Analysis Of Differential Expression Proteins Between Attenuated Strain And Virulent Strain Of Brucella Abortus

Brucellosis is a zoonotic disease caused by the genus Brucella. It is widely popular in the world and affects the development of the healthy and sustainable livestock; Humans is infected by touching infected animal secretions or eating infected animal products, so it is serious threat to human health and public safety. Brucella can infect embryonic trophoblast cells, then cause miscarriage,but the molecular pathogenesis is unclear.There are some differences in own growth,reproduction and infection of the host cell,and induce the apoptosis between the strength of the Brucella strains.Therefore,the study on differentially expressed proteins of the strength of the Brucella strains is great significance.Objective:The study based on the difference protein (dsbG, ccrB, rirA, vceA) obtained by contrasting Brucella virulent strain2308and vaccine strains RB51.Then we constructed the corresponding gene deletion strains,and compared on the growth conditions and the ability of intracellular survival,as well as apoptosis of infected cells.It can explore the function of these differentially expressed proteins which reveal the molecular pathogenesis of Brucella and lay the foundation for the development of new drugs.Methods:(1) Brucella S2308was heat-inactivated which act as template. Upstream and downstream homology of dsbG、ccrB、rirA and vceA gene were High-fidelity amplificated, and then homology and kanamycin resistance gene were fusioned by fusion PCR technology. The fusion fragment directly connected to the pMD19-T cloning vector,which was transfected into Brucella S2308. Screening and testing the genetic stability on S2308ΔdsbG, ΔccrB, ΔrirA and ΔvceA gene deletion strains,we draw Brucella growth curve and observed the aggregation of the bacteria in the stationary culture.(2) HPT-8cells were infected by Brucella parent strain and S2308ΔdsbG, ΔccrB, ΔrirA, ΔvceA gene deletion strains, and then observed its morphology and analysised intracellular survival capacity.(3) Nucleus and DNA fragmentation of apoptotic cells were analysised by Hoechst staining and DNA ladder, the apoptosis rate were detected by flow cytometry.Results:(1) we build and screened Brucella S2308ΔdsbG, ΔccrB, ΔrirA and ΔvceA gene deletion mutant, and reverse mutation does not occur within10generations. Growth state in vitro of the deletion mutant and parent strain2308is similar that bacteria enter the logarithmic growth phase by32h, plateau by40h, but the concentration of bacteria is different in difference time points. CdsbGared Brucella ΔrirA and ΔvceA gene deletion strains with the parental strain, the plateau time cut down4h; Static culture of Brucella gene deletion strains showed turbid growth, it is no significant change cdsbGared with the parent strain2308;(2) the Brucella S2308ΔdsbG, ΔccrB ΔrirA and ΔvceA deletions strains infected trophoblast cells (HPT-8).The cells became round、shrinkage,wider on cell interval and esay to appear polymerization;Results of cells adhesing Brucella show that the number of bacteria entering the cells increase and reach saturation after90min;On the same time, the number of ΔrirA bacteria was significantly more than the parent strain,and the number of AdsbG bacteria was significantly less than the parent strain. CdsbGared the parent strain2308, the the number of intracellular ΔccrB and ΔvceA deletion mutant is slightly increased, but t intracellular bacteria is no significant difference after90min.Results of Brucella intracellular survival experimental show that four hours after infection, intracellular bacteria (Brucella abortus RB51,ΔccrB and ΔvceA deletion mutant)continued to decline. But the overall trend is consistent between S2308ΔdsbG deficient mutant and the parent strain, the intracellular amount of bacteria of ΔrirA deficient mutant is basically remained at a certain level.(3) HPT-8cells were infected by Brucella(MOI200) at different time points. The apoptosis rate is found by flow cytometry.We found apoptosis rate increased with the extension of the infection time. CdsbGared with the parental strain2308, the apoptosis rate of the ΔccrB and ΔdsbG gene deletion strains is no significant change,but the apoptosis rate of ΔrirA and ΔvceA strain was increased. HPT-8cells were infected by Brucella(MOI500) at different time points. With the extension of the infection time, The Hoechst fluorescent staining increased significantly on the nuclear shrinkage and edge set, Using the apoptotic DNA ladder extraction kit,we analysis the DNA fragments of apoptotic cells and found electrophoretic bands show as gradient strip after infection6days.Conclusion:We constructed and obtained S2308ΔdsbG,ΔccrB, ArirA and ΔvceA gene deletion strains.and its growth trend is simila with Brucella2308. but the growth rate of the ArirA and ΔvceA gene deletion strains is significantly faster in Brucella2308; Lacking of the genestrains,their aggregation can not change. After deleting the dsbG, ccrB, rirA and vceA gene, Brucella intracellular survival phenotype changes. The ability to infect and intracellular fecundity of the ΔrirA deletion mutant were significantly higher than the parental strain, but ΔdsbG gene deletion strains is less.Compared with the parent strain, the reproductive capacity of gene deletion strains for ΔccrB and ΔvceA is lower. The study found that ΔrirA and ΔvceA deletion mutant can promote the apoptosis of cell. This study provides clues to reveal the molecular mechanisms of Brucella miscarriage, and promote the research and development of new drug.