Expression Of EF/MRP Antigen Fragment Of Streptococcus Suis Serotype 2 In Lactic Acid Bacteria And Its Oral Vaccination

Streptococcus suis serotype 2(SS2) is the important pathogen of diseases in pigs.It is a major cause of meningitis,septicemia,arthritis and bronchopneumonia in young pigs or even meningitis in humans.Major virulence factors have been recognized in recent years,including extracellular protein factor(EF),muramidase released protein(MRP),suilysin(SLY) etc.Since lactic acid bacteria(LAB) are generally regarded as safe(GRAS) and can act as a live delivery vehicle,to develop an oral vaccine to protect from SS2,the EF/MRP antigen fragments were expressed in LAB for the first time in this study.The epf/mrp gene fragments were cloned and ligated with promoter P59 and signal peptide sp_Usp45, thus EF constitutive secretary expressive vector pHL301 and MRP constitutive secretary expressive vector pHL302 were constructed respectively.Similarly,the PCR- amplified epf/mrp gene were ligated with promoter P_nisz and signal peptide sp_Usp45,generating nisin-controlled EF protein expression vector pHL401 and MRP protein expression vector pHL402.At the same time,epf and mrp gene were fused together to get the EF-MRP constitutive expression vector pHL500 and nisin-controlled expression vector pHL600.Then,the constitutive vector pHL301 and pHL302 were transformed into Lactococcus lactis MG1363,Lactobacillus casei ATCC27092,Lactobacillus brevis ATCC8287,Lactobacillus acidophillus HLA and Lactobacillus plantarum NCIMB8826,respectively.The nisin-controlled vector pHL401,pHL402 and pHL600 were transformed into L.lactis NZ9000,respectively. And pHL500 were transformed into Lb.plantarum NCIMB8826.Western blotting analysis demonstrated that the protein products(EF/MRP/EF-MRP) were expressed in the cytoplasm or secreted into the medium.In particular,the EF or MRP protein can be better expressed during the whole growth phase of recombinant Lb.casei ATCC27092.The expression product targeted in supernatant while little found in the cytoplasm in recombinant Lb.plantarum NCIMB8826.Furthermore,the expression product can still be detected in exponential late phase when OD₆₀₀ of incubation reached 1.6.The higher expression level emerged in the recombinant L.lactis NZ9000 by nisin induction.The recombinant strain Lb.casei ATCC27092,Lb.plantarum NCIMB8826 and L. lactis NZ9000 were administered to C57BL/6J mice orally.Results showed that orally delivered recombinant Lb.casei ATCC27092/pHL301 can induce significant EF-specific IgG in the sera and recombinant Lb.casei ATCC27092/pHL302 can also induce significant MRP-specific IgG in the sera.But the specific IgG was lower in recombinant Lb.plantarum NCIMB8826 and L.lactis NZ9000 administered mice. These results suggest that recombinant strain Lb.casei ATCC27092/pHL301 and Lb. casei ATCC27092/pHL302 may be applicable as an oral vaccine to induce protective immunity against SS2 in C57BL/6J mice.