Expression,Immune Effect And Biology Characteristics Of Three Different EAG Mutants Of Streptococcus Equi Subspecies Equi

Strangles is a highly infectious disease characterized by nasopharyngeal symptoms of equine and caused by Streptococcus equi subspecies equi(S.equi).The disease can lead death of horses,affects the development of horse industry worldwide,and has caused serious economic losses to horse breeding industry in Xinjiang for the past few years.EAG protein was an important IgG binding protein,which involved in the immune evasion.Presently there are less research on the variation of EAG and its relationship with the pathogenicity and immunity In order to understand the EAG variation during the epidemics and evaluate the role of EAG in the pathogenesis and immune of S.equi.In this study,the EAG of S.equi were cloned,expressed and these purified EAG rocombinant proteins were immunized on mice and horse.Their immuobiological characteristics were investigated.In this study,three different EAG mutants genes were amplified(EAG5012?EAG823 and EAGHT1112)and prokaryotic expression recombinants were constructed.After induction,SDS-PAGE analysis result indicated expressed recombinants EAG823,EAGHT1112 and EAG5012 proteins were about 25 kDa and Western-blot results showed these recombinant proteins had good immunogenicity.The purified EAG and SeM,IdeE2 proteins were used to immunize mice and horses and after immunization the serum antibody and antibody subtypes of immunized mice and horse were investigated by ELISA.The results showed that the antibody titer of EAG5012 and EAGHT1112 immunization groups(1:72 900)were higher than that of EAG823 immune group(1:24 300).Antibody titers of the SeM+IdeE2+EAG5012 immune group was 1:218 700.In addition,the antibody type of all immune group mainly were IgGl and IgG3,and IgG1 was the highest.The immunized mice were challenged with S.equi 5012 and the bacteria loads in the tissues of heart,liver,spleen,lung,kidney and the histopathology observation was detected.The results showed that the challenge protection rate of EAG5012 immunization group was 86.7%higher than that of EAGHT1112 immunization group(80%)and that of EAG823 immunization group(66.7%)and the protection rate of SeM,IdeE2 and EAG5012 combined immunization group was 87%.The bacterial load results indicated that EAG5012 immunization group had less bacterial load than the EAG823 and the EAGHT1112 immunization group,The bacterial load of SeM,IdeE2 and EAG5012 immunization group was less than that of other group immunized with each protein(P<0.05).The SeM,IdeE2 and EAG5012 immuized group has the better immunization effect.Mouse spleen lymphocytes were used to detect the activation and proliferation of lymphocytes of each immunization group.The results showed that the proliferation rate of spleen lymphocytes of EAG5012 immunization group was 150%,significantly higher than EAG823 immunization group(120%).SeM+IdeE2+EAG5012 immunization group had the highest stimulation rate(160%),significantly higher than individual immunization group.After immunization and challenge,the cytokines expression levels of IFN-?,IL-2,IL-4,IL-6,IL-10,IL-12 and IL-17A in mice serum and the expression level of IFN-y,IL-4,IL-6 and IL-10 in horse serum were tested by ELISA method.The results showed that the expression level of these cytokines in mice serum and horses serum increased significantly,expression level of these cytokines in EAG5012 immunization group was significantly higher than EAG823 and EAGHT1112 immunization group.The cytokine expression level of mice and horse in SeM+IdeE2+EAG5012 immunization group were higher than those of each protein alone immunization group.The results of phagocytic experiment on horse polymorphonuclear neutrophils showed the inhibitory effect of EAG5012 protein was higher than those of EAG823 and EAGHT1112 and the inhibitory effect of EAGHT1112 protein was higher than that of EAG823(P<0.05).The inhibitory effect was positively correlated with protein concentration.The inhibitory effect of SeM+IdeE2+EAG5012 protein was higher than that of each protein.The result of opsono-phagocytosis of these protein-specific antibodies on equine PMN showed that opsono-phagocytosis of the EAG5012 specific antibodies was higher than that of EAG823 and EAGHT1112 antibodies,opsono-phagocytosis of SeM+IdeE2+EAG5012 specific antibody was significantly better than their individual protein specific antibodies.In summary,the three different EAG mutants of S.equi have distinct immunobiological characteristics.SeM,IdeE2,and EAG5012 immunized incombination with mice and horse had better immune effects.The study will provides experimental basis for vaccine research and prevention and control of equine strangle infection.