| Porcine epidemic diarrhea(PED)is an alimentary canal contagion that is induced by porcine epidemic diarrhea virus(PEDV)in pigs of different ages.Mucosal infection and mucosal immunity are prominent features of the disease,and the design of a vaccine targeting mucosal immunity is important for the scientific prevention and control of the disease.In this study,a comparative study on the level of immune response induced by PEDV S1 immunoprotective antigen expressed by different lactic acid bacteria was conducted to provide a basis for the selection of suitable lactic acid bacteria as receptor vectors,using the characteristics of intestinal mucosal immunity induced by heterologous antigen expressed by lactic acid bacteria.The main research results are as follows:Lactobacillus casei(L.casei 393),Lactobacillus paracasei(L.paracasei 27-2),Lactobacillus royale(L.reuteri J31)and Lactobacillus johnsonii(L.johnsonii 6332)were used as hosts to construct four recombinant strains expressing PEDV S1 protein,p PG-T7g10-S1/L.casei 393,p PG-T7g10-S1/L.paracasei 27-2,p PG-T7g10-S1/L.reuteri J31 and p PG-T7g10-S1/L.johnsonii 6332.Western Blot and indirect immunofluorescence were used to identify the expression of S1 protein and the results showed that all four recombinant strains expressed S1 protein of approximately 99 k Da.The probiotic properties,resistance and genetic stability of the four recombinant Lactobacillus strains in relation to immunity were investigated in depth.The results showed that the p PG-T7g10-S1 recombinant Lactobacillus plasmid transformed four strains of Lactobacillus and expressed exogenous proteins stably after multiple passages without plasmid loss.All four strains had good colonisation characteristics in the pig intestine,with the recombinant bacterium p PG-T7g10-S1/L.paracasei 27-2 having a better colonisation capacity.In order to compare the level of vaccination of neonatal piglets with four strains of the recombinant bacillus,this study firstly investigated the difference in the effect of two different immunization procedures,and showed that the recombinant bacteria p PG-T7g10-S1/L.reuteri J31 induced humoral and mucosal immunity effectively in piglets under both immunization procedures.Compared with three consecutive immunizations,the immunization program of one postnatal immunization showed more significant levels of serum-specific Ig G and mucosal-specific SIg A in newborn piglets(P<0.05).The levels of serum antibodies,mucosal antibodies and cytokines induced by oral immunization of piglets with four strains of recombinant Lactobacillus using this immunization program were compared.The results showed that oral immunization of piglets with all four recombinant Lactobacillus strains could induce specific immune responses.The specific Ig G level of recombinant p PG-T7g10-S1/L.Paracasei 27-2 group was significantly higher than that of p PG-T7g10-S1/L.Johnson II 6332 group(P<0.05).The specific SIg A was also significantly higher than that of the other groups(P<0.05),and the specific Ig G and SIg A produced by the two groups had neutralizing activity against PEDV.No significant differences were observed in the serum levels of cytokines IFN-γ,IL-2,IL-4 and IL-10 after immunization of piglets with the four recombinant strains;however,the levels of cytokines IL-4,IL-6,IL-21,TNF-β,IL-17 and APRIL in the jejunal mucosa of piglets were significantly higher in the p PG-T7g10-S1/L.paracasei 27-2group than in the other groups(P<0.05).In summary,in this study,the PEDV S1 gene was inserted into the Lactobacillus constitutive expression plasmid vector,and different Lactobacillus species were transformed and immunised in newborn piglets,and the levels of immune response induced by different Lactobacillus species were compared.The immune response of the recombinant L.paracasei 27-2 was significantly better than that of the other three recombinant strains.The results of this experiment provide scientific data for the construction of a more effective oral vaccine for Lactobacillus. |
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