| Mastitis was one of the highest infectious diseases for dairy industry and it was difficult to control.This disease cause serious economic loss and seriously threat people's health.Staphylococcus aureus was important pathogen that cause mastitis in dairy cows.This pathogen secreted many virulence factors,possess antibiotic resistance character of immune evasion.EsxB was an secreted proteins of S.aureus and Play important role in it's infection process and deep abscesses as an secreted proteins of S.aureus.At present function research of EsxB is very limited.The aim of the study is to elucidate the immunobiological features of EsxB.In this study,we amplified EsxB gene and constructed its prokaryotic recombinant plasmid.The expressed fusion protein was purified and was used to immunize mice.The immune characteristics of EsxB was investigated.To further evaluate the immune of EsxB.The EsxB protein and 5 proteins: Esx A,Ef B,Clf A,Fli C,Flj B previously expressed by our laboratory were used to immunize mice,the serum antibody level titers and antibody subtypes were detected by ELISA.SDS-PAGE and Western-blot results showed that molecular weight of EsxB recombinant protein is 17.6 k Da.The antibody titer in EsxB immunization group could reach 1:72900 and higher than that of other groups(P<0.05).The antibody subtype of immunized mice was mainly Ig G1 and Ig G2 b.The antibody level of EsxB+Esx A immunization group was higher than EsxB+Flj B,EsxB+Fli C and EsxB+Esx A+Clf A.This result indicatde that EsxB and Esx A+EsxB protein could stimulate butter humoral immune response in mice.After immunization bacteria loading of tissue(heart,liver,spleen,lung and kidney).histopathological observation and challenge protection of immunized mice were detected.The result showed that EsxB immunization groups tissue bacteria loading obviously lower than Fli C,Flj B,Ef B immunization groups and EsxB+Esx A+Clf A immunization group was the lowest.Histopathological observation showed that S.aureus GW(20-2)challenge groups showed obvious pathological changes in liver,spleen,lung and kidney of mice could had.Hyperemia,hemorrhages,inflammatory cell infiltration and tissue degeneration.these lesions significantly reduce and improved in Esx A+EsxB immunization group.protection rate of EsxB immunization groups was 75% higher than that of Ef B.protection rate of Esx A+EsxB immunization group was 100% higher than that of Esx A 87.5% and EsxB 75%,protection rate of Esx A+EsxB+Clf A immunization groups was 100%.The result also showed that Esx A+EsxB and Esx A+EsxB+Clf A could provide mice better protection from the S.aureus infection.Then the expression levels of TLRs,MHC-?,RP105,TCR and IL-10 Immune cell subtype were test by Real-time PCR and flow cytometry.our real-time result showed that,the m RNA expression levels of TLR2,TLR3,TLR4,MHC-?,RP105,TCR and IL-10 were significantly increased and TLR2,TLR3,TLR4 of combination immunization groups were higher than single immune groups(P<0.05).The IL-10 and immune ralated of EsxB immunization group were lower than that of other groups.TLR2,TLR3,TLR4,MHC-?,RP105 and TCR of Esx A+EsxB immunization groups were higher than that of other immunization groups,EsxB+Esx A+Clf A immunization group IL-10 were significantly higher than others.The flow cytometry detect Immune cell of our result showed that all immunization groups CD4 expression level was increased CD4,CD8 and CD69 expression level of Esx A and Esx A+EsxB were higher than EsxB immunization group.The study will lay a foundation for the controlling and prevention of bovine mastitis. |
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