Expression And Comparison Of Immune Effect Of Two Different Mutants SrtA Of Streptococcus Equi Subsp.equi

Strangles is a cute and contagions infectious disease of horse,which is characterised by abscessation of the lymph nodes of the head and neck and seriously could cause death of horse.The disease has caused tremendous economic losses of the equine industry in the world.Strangles mainly caused by Streptococcus equi subsp.equi（S.equi）.SrtA is an important virulence protein of S.equi and highly conserved.SrtA is the pathogen of strangle and recognizeing secreted proteins containing an LPXTG motif and covalently attaches these proteins to cell wall peptidoglycan.As an important horse breeding area,Xinjiang horse industry has been seriously affected by the disease with the horse breeding expanded.In this study,SrtA（750 bp）was amplified S.equi isolation in Xinjiang and prokaryotic expression recombinant was constructed.The expressed protein was detected by SDS-PAGE and Western blot.The results showed that p ET-28a-SrtA-2,SrtA-3 were successfully constructed,the SrtA-2,SrtA-3 was induced exprssed and purified,SDS-PAGE result showed that the molecular weight of the protein is approximately 27 k Da,and its immunogenicity was confirmed by Western blot.The purified SrtA and M-like proteins（SeM）,Immunoglobulin-binding proteins（EAG）previously purified exprssed by our laboratory were used single and incombination to immunized mice.The serum antibody titer and antibody svbtype of immunized mice were detected.Our results showed that the antibody titer of SrtA after 2^ndimmunization reached 1:13500,the antibody titer of SeM and EAG were 1:13500,and the antibody titer of the SeM+EAG+SrtA-3 reached 1:72900.Which was higher than the SeM+SrtA-3,EAG+SrtA-3 immunization group and other immunization group.Antibody type result showed that Ig G1 and Ig G2b antibodies were the main.The results demonstrated that SrtA,SeM and EAG proteins elicited Th2-type in mice.After immunization and challenge,bacterial burden,histopathology,and immunoprotective capacity were measured.Our result of bacterial burden and histopathology showed that SrtA-3immunization group had better protection in liver and lung than SrtA-2,SrtA-2 immunization group had better protection heart,spleen,and kidney than SrtA-3.The bacteria burden and histopathological changes observation of in the organ of EAG+SeM+SrtA-3 was the best than those of other groups.Mouse challenge results showed that survival rate of SrtA-2 was 87.5%,survival rate of SrtA-3 was 75%,for EAG+SeM+SrtA-3 immunization group was 87.5%,for SeM immunization group was 62.5%.To test the protection of against 5 differ S.equi strains（ZZM-2,ZZM-3,5012,84-3,ZTT-MY-1）survival rate range 50%to 80%,against S.zooepidemicus strains ZTT-SY-1 and MXH-SY-1 survival rate was only 25%.Attacked bacteria mice after weight loss,4-5d weight loss to a minimum,9-10 d recovery before weight,EAG+SeM+SrtA-3 group is better than other immunization groups.Lymphocytes cytokines and immune related molecules of immunization mice were detected using flow cytometry and real-time PCR.The results showed that SrtA could induce mice produceing CD4⁺T,CD8⁺T,CD69⁺T cells.The expression level of CD4⁺T and CD8⁺T cells inSrtA-2 immunization group was higher than SrtA-3 group,and the expression level of CD69⁺T cells was lower than SrtA-3 group.The real-time PCR results showed that the expression level of IL-10and RP105 of SrtA-3 immunization group was higher than that in SrtA-2 group.The expression level of MHC I,TCR,TLR2,TLR3 and TLR4 in SrtA-2 immunization group was higher than SrtA-3 group;the expression level of IL-10 in EAG immunization group was the highest than SrtA-3,EAG,SeM,the expression level of MHC I,TCR,TLR2,TLR4 in SrtA-3 immunization group was the highest than other groups,the expression level of RP105 in SeM+SrtA-3 immunization group was the highest than other groups,the expression level of TLR3 in SrtA-3 and EAG+SeM+SrtA-3immunization group was the highest than other groups.In this study,different mutants of SrtA were expressed.It was found that the immune effect of SrtA-2 was better than that of SrtA-3 in mouse models;mice immunized with different proteins EAG,SeM,and SrtA-3,SeM+EAG+SrtA-3 immunization group had the best protection effect.The results of our study provide experimental data for the prevention of horse strangles.