Recombinant Bacillus Subtilis Spores Expressing Cholera Toxin B And Ovalbumin Protein Prevent Against OVA-induced Food Allergy In Mice

Objective:The prevalence of egg allergy is increasing year by year.Ovalbumin(OVA)is the primary allergen in eggs.The combination of immune tolerancic adjuvant cholera toxin B(CTB)and allergen can effectively prevent food allergy.In this study,CTB was fused with OVA and expressed on the surface of Bacillus subtilis to construct the novel allergen vaccine.OVA-induced food model was built to study the prevent effect and machnism of CTB-OVA Bacillus subtilis spores in mice.It provides a new insight and novel strategy for the development of allergen vaccine and the immunotherapy of food allergy.Method:Construction of recombinant B.s-Cot C-CTB-OVA Bacillus subtilis:OVA gene were synthesized and amplify by PCR with specific primers.Then OVA gene was subcloned into p ET28a CTB plasmid.CTB-OVA gene was amplified by PCR with p ET28a-CTB-OVA plasmid as templated,then cloned into p US186-Cot C plasmid to construct p US186-Cot C-CTB-OVA plasmid.Then p US186-Cot C-CTB-OVA plasmid was identified by PCR and sequencing and transformed into Bacillus subtilis WB600.Spores were induced by DSM nutrient depletion method.SDS-PAGE and Western blot were used to identify the expression of recombinant CTB-OVA protein.Prevention strategy of OVA food allergy by oral administration of recombinant B.subtilis spores:BALB/c mice were randomly divided into 5 groups:Naive group,food allergy model group,B.s-Cot C spore group,B.s-Cot C-CTB spore group and B.s-Cot C-CTB-OVA spore group.Four weeks before the establishment of OVA food allergy model(-week 4--week1),10~9 recombinant B.subtilis spores were oral administrated for four weeks,three times a week.On day-1,feces were collected for OVA-specific s Ig A detection,16S r DNA sequencing and fecal microbiota transplantation.On day 0 and 14,mice were intraperitoneal injected with 50μg OVA and 1mg Alum except for the naive group.Then the mice were boosted with 50mg OVA every other day on the 18th,20th,22th and 24th day.On the 28th day,the mice were challenged with 100mg OVA and the diarrhea and allergic symptoms score were recorded.The serum of mice was collected for the detection of immunoglobulin(Ig E,Ig G1,Ig G2a).The spleen cells of mice were isolated for the detection of Treg cells,mast cells and cytokines.Study on recombinant spores therapy for fecal microbita transplantation(FMT)in mice:FMT suspension was prepared by collecting the feces of mice with oral B.s-Cot C and B.s-Cot C-CTB-OVA spores before the establishment of OVA food allergy model(day-1).BALB/c mice were randomly divided into four groups:Naive group,food allergy model group,B.s-Cot C FMT group and B.s-Cot C-CTB-OVA FMT group.Before fecal bacteria transplantation,B.s-Cot C FMT group and B.s-Cot C-CTB-OVA FMT group were treated with antibiotics.The mice were transplanted with 1/500 fecal bacteria of their own weight for 7 consecutive days.Then food allergy model was established and food allergy symptoms and diarrhea score were recorded and serum OVA-Ig E was detected.Results:PCR and sequencing results showed that 1482 bp CTB-OVA gene was successfully inserted into the open reading frame of p US186-Cot C plasmid.SDS-PAGE and Western blot showed that the recombinant B.s-Cot C-CTB-OVA spores had a clear expression band near 42.9 Kda size.In this experiment,the recombinant B.s-Cot C-CTB-OVA was successfully constructed and expressed on the spore surface after induction by DSM spore medium.Compared with other groups,oral recombinant CTB-OVA spores could significantly increase fecal OVA-s Ig A(p<0.05).After 100 mg OVA challenge,oral administration of recombinant B.s-Cot C-CTB-OVA spores could alleviate the food allergy symptoms(p<0.05),diarrhea symptoms(p<0.05)and diarrhea rate(p<0.05),compared to the allergy model group.ELISA and flow cytometry showed that the levels of OVA-Ig E,Ig E~+plasma and OVA-Ig G2a in B.s-Cot C-CTB-OVA group and B.s-Cot C-CTB group were significantly lower than allergy model group and B.s-Cot C group(p<0.01).In addition,OVA-Ig G1 in B.S-Cot C-CTB-OVA group was significantly lower than allergy model group(p<0.05).Compared with B.s-Cot C and allergy model groups,B.s-Cot C-CTB-OVA and B.s-Cot C-CTB spores could down-regulate IL-4(p<0.05).and increase IL-10(p<0.05)in spleen.Compared with allergy model group,B.s-Cot C group and B.s-Cot C-CTB group,oral administration of B.s-Cot C-CTB-OVA spores could inhibit spleen c-kit~+Ig E~+mast cells(p<0.001)and CD11b+singlecf+eosinophils(p<0.01).Compared with allergy model group,the number of Treg in B.s-Cot C-CTB-OVA and B.s-Cot C-CTB groups was significantly increased(p<0.05)and the the number of Treg in B.s-Cot C-CTB-OVA group was higher(p<0.05).16S rDNA sequence showed that B.s-Cot C,B.s-Cot C-CTB and B.s-Cot C-CTB-OVA spores could change the composition of intestinal microbita.Oral administration of B.s-Cot C-CTB-OVA spores could up-regulate the abundance of bacteroidia and down-regulate the abundance of Firmicutes and Epsilonbacteraeota.In Family level,B.s-Cot C-CTB-OVA spores could up-regulate the abundance of Lactobacillus lacteae and Muribaculaceae.B.s-Cot C-CTB-OVA spores could also up-regulated the abundance of odoribacter at the Genus levels.FMT showed that:after treatment with B.s-Cot C-CTB-OVA FMT and B.s-Cot C FMT,the allergic symptoms and diarrhea symptoms of mice were not significantly changed.However,B.s-Cot C-CTB-OVA FMT mice could significantly reduce the level of OVA-Ig E in serum(p<0.05).It is suggested that the changes of intestinal microbiota induced by B.s-Cot C-CTB-OVA may play a role in the inhibition of OVA food allergy.Conclusion:Oral administration of recombinant B.s-Cot C-CTB and B.s-Cot C-CTB-OVA spores can alleviate the symptoms of food allergy and diarrhea,reduce serum Ig E and increase the number of Treg in OVA food allergy mice.In addition,B.s-Cot C-CTB-OVA can effectively induce the production of s Ig A and inhibit the number of mast cells and eosinophils.At the same time,B.s-Cot C-CTB-OVA can change the composition of intestinal microbiota and increase the abundance of Lactobacillus and Muribaculaceae.Moreover,the recombinant CTB-OVA can inhibit the OVA-Ig E of recipient mice by FMT.In conclusion,oral administration of recombinant CTB-OVA Bacillus subtilis can inhibit OVA food allergy in mice,and the mechanism is related to the changes of intestinal microbiota.