Characterization And Immunological Evaluation Of Recombinant Lactobacillus Expressing APEC FimA-ompC Fusion Protein With Targeting Peptide Of Microfold And Dendritic Cells

Avian colibacillosis caused by avian pathogenic Escherichia coli(APEC)can lead to a large number of deaths in embryo and chicken with the main clinical symptom such as fatal acute enteritis,peritonitis,salpingitis,subacute pericarditis,E.coli granuloma and sepsis.Avian colibacillosis is a common disease in chicken,resulting in enormous economic losses to the poultry industry worldwide.There are many serotypes of pathogenic E.coli.Among them,O1,02 and 078 are the main serotypes causing avian colibacillosis.APEC can cause systemic infection through respiratory tract and gastrointestinal tract.At the same time,because of the many serotypes and complex antigen structure of APEC,it is difficult to prevent and control avian colibacillosis.APEC infection mainly occurs through intestine,so it is an effective way to resist APEC infection by stimulating the mucosal immune system of the intestinal mucosal tissue.In this study,we use lactic acid bacteria as immune antigen delivery carrier to construct recombinant lactic acid bacteria system of targeting intestinal micro fold cells(M cells)and dendritic cells(DC)to delivery fimA and ompC protein of APEC,and the feasibility of using genetic engineering Lactobacillus via oral immunization to induce mucosal immunity against APEC infection was explored.There were 8 strains of lactic acid bacteria strains isolated from the intestinal tract of chicken,and colonization ability test results showed that the colonization ability of L.saerimneri(M11)was better than that of other strains.Moreover,the probiotics of L.saerimneri(M11)was better than that of Lactobacillus casei that was commonly used as vaccine antigen delivery carrier.Therefore,L.saerimneri(M11)strain was used as an immune antigen delivery carrier to construct genetic engineering Lactobacillus in this study.Using L.saerimneri M11 strain as antigen delivery carrier to deliver fimA and ompC protein of APEC combined with DC targeting peptide DCpep and M cell targeting peptide Co1,targeting delivery recombinant L.saerimneri DC(M11)system pPG-T7g110-PPT-OmpC-fimA-Col-DCpep/M-11(pPG-OF-Col-DCpep/M11)and non targeting recombinant L.saerimneri(M11)pPG-T7g10-PPT-OmpC-fimA/M-11(pPG-OF/M11)was constructed with the constitutive expression vector pPG-T7g10-PPT,respectively.The tolerance to the environmental performance of the digestive tract and colonization ability of recombinant pPG-OF-Co1-DCpep/Ml 1 and pPG-OF/M11 were evaluated,and results showed that the recombinant Lactobacillus pPG-OF-Col-DCpep/M11 and pPG-OF/M11 had good digestive environment tolerance and intestinal colonization ability.Following the culture of the recombinant pPG-OF-Col-DCpep/Mll and pPG-OF/M11 in MRS medium at 37? for 16 h,the target proteins were constitutively expressed identified by Western blot assay.In this study,the ability of recombinant pPG-OF-Col-DCpep/M11 and pPG-OF/M11 to regulate the maturation of chicken bone marrow DCs was evaluated by MLR method,and results showed that the stimulation index of pPG-OF-Col-DCpep/M-11 was higher than that of pPG-OF/M-11 and pPG/M-11(p<0.05).Meanwhile,the immunogenicity in SPF chicks of recombinant Lactobacillus pPG-OF-Col-DCpep/M11 and pPG-OF/M11 was systematically evaluated via oral immunization.The SPF chicks were randomly divided into 4 groups(35 chicks for each group):pPG-OF-Col-DCpep/M11(experimental group),pPG-OF/M11(experimental group),M11 group and PBS group(control groups).Immunization program was as follows:the chicks were orally administered on three consecutive days(days 1,2,and 3)and a booster immunization was administered on days 14,15,and 16.For the detection of specific IgG and IgA levels by ELISA,stool samples were collected at days 0,4,7,10,14,17,21,28,and 35 after immunization,and sera,cecum lavage and nasal lavage samples were collected at days 0,7,14,21,and 35 after immunization,respectively.And then,the immunized chicks were challenged with E.coli CVCC1553 to evaluate the immune protective effect of recombinant Lactobacillus pPG-OF-Co1-DCpep/M11 and pPG-OF/M11.The results showed that:compared with the control group,significant level of anti-fimA specific IgA/IgG antibody and anti-ompC specific IgA/IgG antibody(p<0.01)were induced in SPF chicks orally immunized with recombinant lactic acid bacteria pPG-OF-Co1-DCpep/M11 and pPG-OF/M11;compared between recombinant Lactobacillus pPG-OF-Col-DCpep/M11 and pPG-OF/M11,on days 4 after the first immunization,the mucosal IgA antibody levels in stool samples of immunized chicks with pPG-OF-Col-DCpep/M11 were higher than that of immunized chicks with pPG-OF/M11,and on days 7 after the first immunization,the specific IgA/IgG antibody levels induced in chicks orally immunized with pPG-OF-Co1-DCpep/M11 were significantly higher than that induced in chicks orally immunized with pPG-OF/M11(p<0.05).The challenge experiment results showed that the protection rate of recombinant pPG-OF-Co1-DCpep/M11 was 80%and the protection rate of recombinant pPG-OF/M11 was 70%.Our data indicated that DCs and M cell targeting delivery recombinant pPG-OF-Col-DCpep/M11 has a better immunogenicity.In summary,the M cells-targeting and DCs-targeting recombinant lactic acid bacteria pPG-OF-Col-DCpep/M11 expressing fimA and ompC antigen of APEC has good immunogenicity which not only can induce local mucosal immune response but also induce humoral immune response,and has good immune protection effect.This suggests that the Lactobacillus delivery system may be a promising strategy for the development of vaccine against APEC infections.