Screening And Evaluation Of Immunogenic Proteins Of Erysipelothrix Rhusiopathiae

Our laboratory researched cell wall proteins of Erysipelothrix rhusiopathiae virulent SE38 and attenuated G4T10 by proteomics.A number proteins of S/G fold in 1.5-2.0 were detected.Some of these proteins has a good immune response with convalescent serum of Erysipelothrix rhusiopathiae.These proteins were researched its ability as a subunit vaccine.ERH₀412,PotA,UgpB,SpaA,ERH₁291,FusA,ERH₁090 and CbpC genes were successfully cloned into the expression vector pET-28a,and ERH₀384,LemA,ERH₁227 genes were successfully cloned into pET-32a expression vector.The protein SpaA and UgpB were expressed in the supernatant and purified by nickel column.The rest of proteins were mainly expressed in inclusion bodies and purified by the urea denaturation-renaturation.BALB/c mice test showed that the capacity of these proteins induced mice to produce antibody is different.ERH₀412,PotA and SpaA is stronger than ERH₁291,ERH₀384,LemA,FusA,ERH₁090 and CbpC.The protective rate of SpaA?90%?,PotA?60%?,ERH₀412?40%?was higher than that of ERH₁291,ERH₀384,LemA,ERH₁090,FusA,and CbpC in mice.Select SpaA and PotA to perform piglet immunity test.Safety evaluation tests of recombinant protein vaccine for piglets show:piglets after vaccination,injection site no disease,the vaccine was well absorbed,no lumps or suppuration occurred,mining normal food,lively,shiny hair,the body temperature increases slightly at second of inoculation and rapidly returned to normal.Indirect ELISA approved that SpaA or PotA can induce piglets to produce the highest titer of specific antibodies in the Second immunity fourteenth days.High levels of antibodies can be continued for three months until the fourth month of the antibody titer valid.Bacterial content of main organs?heart,liver,spleen,lung and kidney?indicate that there was no bacteria in the organization of piglets immuned SpaA subunit vaccine and G4T10 commercial vaccine.A small amount of bacteria were found in liver and spleen of piglets immuned PotA subunit vaccine.While five organs of the negative control piglets contain large amounts of bacteria.It' s the first time to evaluate protective effect of Erysipelothrix rhusiopathiae subunit vaccine in view of histopathology.Compared with the blank group,pathological lesions of piglets immuned vaccines in the heart,liver,lung and kidney is just the infiltration of inflammatory cells.All piglets of four groups have more severe lesions in the spleen.For example,splenic nodule structure disorder,unclear boundaries between red pulp with white pulp,formation round vacuoles.Protein SpaA is truncated expressied after removed signal peptide and C end anchor sequence.Antigenic determinant regions has a little different amino acids with known sequences,but the immune effect is not reduced.The protein is widely distributed in many kinds of serum,and has more advantages than the inactivated and attenuated vaccine with complex components.We first found that PotA is a better immune effect protein.Survival rate of piglets immuned PotA subunit vaccine group were 50%after challgene inoculation with Erysipelothrix rhusiopathiae.Furthermore,bioinformatic analysis,PCR and Western Blotting confirmed that PotA were widely distributed and highly conservative in the Erysipelothrix rhusiopathiae.