Expression And Immunobiological Characteristic Comparison And Analysis Of GAPDH Of Streptococcus Equi Subspecies Equi

Strangles is an acute,contagious and febrile infations disease of equine and it caused by Streptococcus equi subspecies equi(S.equi).The disease is mainly characterized by head and neck abscesses and is a great threat to equine.This disease presently threatens the development of the horse industry.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was an the important virulence factors of S.equi and was rarely studied.In order to explore its immune effect GAPDH of S.equi was expressed,purified and immunized on mice and its immuobiological characteristics was investigated.In this study,two different size fragments of GAPDH gene were amplified and prokaryotic expression recombinants p ET-28a-GAPDH-L and p ET-28a-GAPDH-S were constructed.After induction,SDS-PAGE analysis result indicated that the expressed recombinant proteins were about 36 k D and 45 k Da.The recombinant protein had good immunogenicity analyzed by western-blot.The purified GAPDH-L,GAPDH-S,EAG,Se M were used to immunize mice.The serum antibody level and antibody subtype of immunized mice were invesfigated by ELISA.The result indicated that the antibody titer of the GAPDH-S immunization group was 1:24300 higher than that of GAPDH-L immunization 1:8100.Antibody titer of the EAG+Se M+GAPDH-S immue group(1:217800)was higher than that of EAG+GAPDH-S,Se M+GAPDH-S immunization group(1:72900)and GAPDH-S,EAG,Se M immunization group(1:24300).In addition,the main antibody type mainly were Ig G1 and Ig G2b.Then immunized mice were challenged with S.equi ZZM-3 and the bacteria burden in the tissues of heart,liver,spleen,lung,kidney and the histopathology observation was carried out.The result indicated that the challenge attack survival rate of GAPDH-S immunization group was 87.5%higher than that of GAPDH-L immunization group 75%;EAG+Se M+GAPDH-S immunization group could reach to 100%,higher than that of EAG+GAPDH-S,GAPDH-S immunization group 87.5%and Se M,EAG,Se M+GAPDH-S immunization group 75%.The bacteria burden result showed that GAPDH-S immunization group had less bacteria loading in heart,spleen and lung than GAPDH-L immunization group,GAPDH-L immunized group had less bacteria loading in liver(P<0.05).The bacteria loading of EAG+Se M+GAPDH-S immunization group in liver,spleen,lung and kidney was significantly lower than other immunization groups(P<0.05).The histopathological observation result showed that GAPDH-S immune group had the better protection in liver,spleen and lung than GAPDH-L immune group.Each immune group had protective effect in kidney.EAG+Se M+GAPDH-S immune group had the best protection in liver,spleen,GAPDH-S and EAG+Se M+GAPDH-S immune group had the best protection in lung.To compare the immunoprotective effects of GAPDH-S and Se M protein,mice were immunized with these proteins the challenge survival rates were tested after challege with different bacteria.Our results showed that Se M could provide 75%survival protection after challege with S.equi and GAPDH could provide 50%?87.5%survival protection after challege with seven different kinds bacterion and it turned out a better protective effect.Lymphocytes cytokines and immune related molecules of immunization mice were detected using flow cytometry and real-time PCR.Flow cytometry results showed that the level of CD4~+,CD8~+,CD69~+T cells induced by the GAPDH-S immunization group significantly higher than GAPDH-L immunization group(P<0.05).The real-time PCR results showed that the cytokine expression level of IL-10,TLR2,TLR3,TLR4,TCR,MHC-?and RP105 of each immunization group increased.Expression level of IL-10of GAPDH-L significantly higher than GAPDH-S immunization group(P<0.05).The expression level of IL-10 in EAG immunization group significantly increased than combined immunization group(P<0.05).TLR2,TLR3,TLR4,TCR,MHC-?and RP105 expression level of GAPDH-S significantly higher than GAPDH-L immunization group(P<0.05).The expression level of MHC-?,RP105,TCR,TLR3 of EAG+Se M+GAPDH-S significantly higher than those of other immunization group.The expression level of TLR2 and TLR4 of GAPDH-S immunization group significantly higher than other immunization group.GAPDH had a good immune effect when immunized single or incombination and provide wide and high challenge survival rates.The results provide experimental basis for future development of strangle prevention and control technology.