Construction And Immune Response Of Lactobacillus Plantarum Targeting Dendritic Cells And Expressing African Swine Fever Virus DNApol And P72 Fusion Antigen

African swine fever is a severe infectious disease caused by African swine fever virus(ASFV).Domestic pigs and various wild boars are susceptible,causing huge economic losses to the pig industry.The virus has an icosahedral shape and a huge and complex genome,which brings great difficulties to vaccine development.So far,there is no safe and ideal vaccine for African swine fever.The G1211R gene encodes the viral DNA polymerase(DNA polymerase,DNApol),which is related to viral replication.The p72 protein encoded by the B646L gene is the major capsid protein involved in viral entry into the host.Lactobacillus plantarum(L.plantarum),as a probiotic,can colonize the host’s gut and modulate the host’s immunity.In addition,it is also an excellent foreign protein expressing bacterium that delivers antigens to the host’s gut through oral immunization.The p WCF vector is an inducible E.coli-L.plantarum shuttle expression vector,which is selected by auxotrophy.Dendritic cells(DCs)are important antigen-presenting cells.Dendritic cell-inducing peptide(DCpep)is composed of 12 amino acids and can specifically bind to DCs.Therefore,in this research,truncated DNA polymerase and partial protein of p72 were selected to construct new functional L.plantarum targeting dendritic cells with non-antibiotic resistance,named as NC8Δ-p WCF-DNApol、NC8Δ-p WCF-p72和NC8Δ-p WCF-DNApol-p72.After induction,they were able to target dendritic cells and anchor and express foreign antigens on the surface of L.plantarum NC8/Δalr.In addition,the immune effect of L.plantarum expressing the fusion antigen of ASFV was evaluated by immunizing mice orally.This research is divided into two parts:(1)Construction and expression verification of new functional L.plantarum expressing ASFV fusion antigenThe ASFV-SY18 gene sequence(MH766894.1)was obtained from the NCBI website.The DNA polymerase was analyzed by DNAstar Protean software and truncated.The 4 epitopes of p72 protein were selected and 3 sets of repeats were made.Separately,they were fused to 3repeated DCpep sequences.These two groups of target gene are fused together to form a co-expressed target gene expressing two antigens.The three groups of target gene were optimized and cloned into p WCF vector.The recombinant plasmid was transferred into asd gene-deficient E.coliχ6212,and the recombinant plasmid was successfully constructed after restriction enzyme digestion verification,polymerase chain reaction(PCR)verification and plasmid sequencing.Electrotransformation into the alr gene-deficient L.plantarum NC8/Δalr,and the successful transformation of the recombinant plasmid was verified by PCR.A white rabbit was immunized with DCpep polypeptide twice,and the serum was collected.The successful preparation of DCpep polyclonal antibody was verified by Western Blot.New functional L.plantarum were incubated with DCpep polyclonal antibody and FITC-labeled anti-rabbit secondary antibody,and detected by flow cytometry.Compared with the empty vector(p WCF)L.plantarum,the new functional L.plantarum had a significant signal deviation,indicating that the foreign protein was successfully expressed.The bacterial solution was incubated with the polyclonal antibody and FITC-labeled secondary antibody,and the new functional L.plantarum showed green fluorescence under a fluorescence microscope,indicating that the foreign protein could be anchored and expressed on the surface of the L.plantarum.The NC8Δ-p WCF-p72 solution was processed by protein sample processing method,and incubated with p72 mouse monoclonal antibody and HRP-labeled anti-mouse secondary antibody.The protein bands with the expected size were verified by Western Blot technology.(2)Study on the immune effect of new functional L.plantarumThe C57BL/6 mice were randomly divided into 5 groups with 10 mice in each group.The PBS and p WCF groups were set as controls,and 200μL PBS and 1.0×10~9 CFU of empty vector L.plantarum(NC8Δ-p WCF)were intragastrically administered,respectively.Each mouse in the new functional L.plantarum group was intragastrically administered with 1.0×10~9 CFU of live bacteria.Initial immunization was performed on 1,3,and 5 days,booster immunization was performed on 15,17,and 19 days,and flow cytometry was performed on the 29th day.The CD80,CD86,and MHC-II costimulatory factors in Peyer’s patches(PPs)were detected.The results showed that oral immune new functional L.plantarum significantly promoted the activation of DCs,and the activation effect of L.plantarum expressing DNApol and p72 was the best.After the cells were co-cultured with antigenic peptides,the expression of IFN-γin CD4~+and CD8~+T cells in the mesenteric lymph nodes(MLNs)of the new functional L.plantarum group was significantly increased;the expression of CD4~+IFN-γ~+and CD4~+IFN-γ~+in spleen cells was also significantly increased.After staining with CFSE,MLNs and spleen cells were co-cultured with antigen peptides for 3 days to measure cell proliferation.The results showed that the number of CD4~+and CD8~+T cells in the MLNs of the experimental groups increased.In the spleen,compared with the PBS group,the co-expression group had a very significant increase in CD4+T cells(P<0.01),and a significant increase in CD8~+T cells(P<0.05).The number of B220~+IgA~+cells in PPs of the new functional L.plantarum groups had an increasing trend,and there was a significant difference between the p72 group and the control group(P<0.05).Two weeks after the booster immunization,the B cell response in the duodenum,jejunum and ileum of mice was detected by immunofluorescence.The number of B220~+IgA~+double positive cells in mouse intestine was observed by fluorescence microscope.In the duodenum and jejunum,there were almost no double-positive cells in the control group,but double-positive cells were clearly seen in the new functional L.plantarum group.The full and partial enlarged images of the cross-section of the ileum showed that the number of double-positive cells in the new functional L.plantarum group was significantly more than that in the control group,indicating that the co-expression group of DNApol and p72 had the best effect.The expression levels of specific s IgA in the feces and specific IgG,IgG1,and IgG2a in the serum were detected of the mice.After 2 weeks of priming and boosting immunization,the s IgA expression level of the experimental groups was significantly increased.Compared with the empty group,the expression levels of specific IgG in the serum of mice fed with new functional L.plantarum was significantly increased after 2 weeks of primary immunization and booster immunization,and 10 weeks after primary immunization(P<0.0001).In 10 weeks after primary immunization,it can maintain a relatively high level.The expression levels of specific IgG1 and IgG2a in serum also reached the highest level after 2 weeks of booster immunization,and the experimental groups was significantly increased compared with the empty group in three periods(P<0.0001).In conclusion,three new functional L.plantarum strains were successfully constructed in this study,which can successfully display the truncated DNA polymerase protein and the partial p72 protein on the surface,and co-express the truncated DNA polymerase protein and the partial protein of p72.Oral immunization of NC8Δ-p WCF-DNApol,NC8Δ-p WCF-p72 and NC8Δ-p WCF-DNApol-p72 enhanced the mucosal,humoral and cellular immunity of mice.Compared with NC8Δ-p WCF-DNApol and NC8Δ-p WCF-p72,NC8Δ-p WCF-DNApol-p72 had the best immunization effect on mice.Therefore,new functional L.plantarum targeting dendritic cells provide a new idea of mucosal immunity for oral live vector vaccine of ASFV,and a new strategy for blocking African swine fever.