| Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),is an acute and highly contagious viral disease characterized by vomiting,watery diarrhea.Pigs of all ages can be infected with PEDV,but it has a high mortality rate in piglets,and is one of the important pathogens causing early death of suckling piglets.PEDV is mainly transmitted by the fecal-oral route,so that oral immunization is the most effective way to prevent and control PEDV infection.Oral immunization of PEDV using intestinal lactic acid bacteria as a live bacterial carrier has both the safety of conventional inactivated vaccines and the persistence of attenuated vaccines.Lactic acid bacteria not only are safe,non-toxic,acid-resistant and bile-resistant,but also can enhance the body’s non-specific immunity and non-specific antibacterial and anti-diarrheal capabilities.Expressing the protective antigen of PEDV in the lactic acid bacteria genome,the foreign protein can be stably expressed without additional inducers,taking full advantages of the mucosal immunity of lactic acid bacteria.In this study,the pyruvate hydratase(enolase,eno)gene locus,the high-efficiency expression site of lactic acid bacteriaΔupp ATCC 393,was used as the insertion site of the lactic acid bacteria genome.The main protective antigen S1 of PEDV was inserted into the genome ofΔupp ATCC393 lactic acid bacteria by double-crossover homologous recombination technology.The transcriptional regulatory sequence of eno was used to initiate the expression of the inserted gene to improve the expression efficiency of exogenous proteins in the lactic acid bacteria genom,so that PEDV S1 gene can be integrated into bacterial chromosomes and replicate stably with the chromosomes.In addition,to compare the differences between the genome and the plasmid expression system of lactic acid bacteria,a recombinant lactic acid bacteria pPG-SD-S1/Δupp ATCC 393 was constructed to express the S1 protein,the main protective antigen of PEDV.Mice and piglets were orally immunized with the recombinant bacteriaΔupp ATCC 393-S1 and pPG-SD-S1/Δupp ATCC 393 to investigate the level of immune response.The main research results are as follows:The fusion fragment of Lactobacillus casei peptidoglycan hydrolase ribosomal binding site and signal peptide sequence and S1 gene(SD-S1 sequence)was obtained to construct pPG-SD-S1recombinant plasmid.The recombinant plasmid was electroporated intoΔupp ATCC 393 to obtain pPG-SD-S1/Δupp ATCC 393 recombinant bacteria expressing S1 protein from the plasmid.The fusion fragment of the upstream homology arm PA,SD-S1 sequence and the downstream homology arm PB was obtained to construct a thermosensitive recombinant vector p GBHCupp-S1.After the recombinant vector was electroporated intoΔupp ATCC 393,theΔupp ATCC 393-S1recombinant bacteria,expressing the S1 protein in genome,was obtained by repeated heating and screening in SDM medium containing 5′-fluorouracil.To evaluate the immunization effect of recombinant Lactobacillus caseiΔupp ATCC 393-S1 and pPG-SD-S1/Δupp ATCC 393,mice were used as animal models for oral immunization.The PBS group andΔupp ATCC 393 group were set as control.The levels of Ig G antibodies in serum and SIg A antibodies in tear fluid,nasal fluid,reproductive tract mucus,feces and intestinal mucus of immunized mice were detected within 70 days of immunization.The results showed that,compared with the control groups,anti-PEDV SIg A and Ig G antibodies in the mice orally immunized with recombinant lactic acid bacteria were significantly increased on the 7th day after immunization(P<0.01),and reaches a peak on the 42nd day after immunization(14 days after the second booster immunization),and then gradually decreased.On the 42nd day of oral immunization with recombinant bacteria,the neutralizing activity of antibodies in the serum of mice was significantly higher than that of the control group(PBS group andΔupp ATCC 393group).Compared with the control group,the proliferation of spleen lymphocytes in the mice immunized orally with recombinant bacteria was significantly increased(P<0.01),in a dose-dependent manner.The secretion levels of cytokines IL-4,IL-2,IL-10,IL-12,IL-17 and IFN-γin the serum of mice immunized orally with recombinant bacteria were significantly higher than those in the control group(P<0.01).After oral immunization of mice with recombinant bacteria,the content of CD4~+T and CD8~+T cells in spleen cells were significantly higher than those in the control group(P<0.01).However,the in vitro neutralization assay,spleen lymphocyte proliferation assay,cytokine level detection,and spleen cell CD4~+T and CD8~+T cell contents were not significant between theΔupp ATCC 393-S1 group and the pPG-SD-S1/Δupp ATCC 393 group.difference.According to the comparison of oral immunogenicity analysis in mice,the more immunogenicΔupp ATCC 393-S1 recombinant bacteria and PBS(control group)were orally immunized to neonatal piglets to evaluate their immune effect.Anti-PEDV specific SIg A levels of piglets’nasal and anal swabs in the oralΔupp ATCC 393-S1 group were significantly rising on day 2 of immunization(P<0.01),with the increase of immunization days.Anti-PEDV-specific Ig G level in the oralΔupp ATCC 393-S1 group was significantly increased(P<0.01)on the 6th day after primary immunization.The neutralizing activity of anti-PEDV Ig G antibody in piglet serum(1:56.32)was significantly higher than that of the control group(P<0.01).On the 6th day after primary immunization,compared with the control group,the levels of IL-4,IL-17 and IFN-γcytokines in the serum of piglets immunized orally withΔupp ATCC 393-S1 recombinant bacteria were significantly increased(P<0.01).To further evaluate the immuned protection effect of oral immunization with recombinant strainΔupp ATCC 393-S1,expressing S1 protein in genome,the challenge experiment was carried out on the 7th day after birth of piglets.2 piglets in the PBS group were given oral 8 m L PEDV ground disease material as the challenge group.To simulate the natural infection conditions,4piglets were orally immunized with PBS as the infection group and 4 piglets were orally immunized withΔupp ATCC 393-S1 as the immuned infection group,co-caged with the challenge group.The remaining piglet orally immunized with PBS was housed alone as the control group.After the challenge,the clinical symptoms of the experimental animals were scored and the nasal and anal swabs were collected,and detected by RT-PCR every day.On the 7th day after challenge,the pathological examination of intestinal tissue,the determination of intestinal viral load and the detection of inflammatory cytokines were performed.Compared with the challenge group and infection group,the clinical symptoms,pathological changes,intestinal viral load and the levels of pro-inflammatory factors were significantly down-regulated in the immune-infection group,but the levels of anti-inflammatory factors were significantly up-regulated(P<0.01).The infection protection rate was 50%,indicating that oral immunizationΔupp ATCC 393-S1 had a certain protective effect on the cohabitation infection of PEDV.the challenge group and infection group,the clinical symptoms,pathological changes,intestinal viral load and the levels of pro-inflammatory factors were significantly down-regulated in the immune-infection group,but the levels of anti-inflammatory factors were significantly up-regulated(P<0.01).The infection protection rate was 50%,indicating that oral immunizationΔupp ATCC 393-S1 had a certain protective effect on the cohabitation infection of PEDV.In summary,in this study,the recombinant lactic acid bacteria pPG-SD-S1/Δupp ATCC 393constitutive expression in plasmid and the recombinant lactic acid bacteriaΔupp ATCC 393-S1constitutive expression in genome were constructed to express PEDV S1 protein.Oral immunization of mice with the two strains of recombinant lactic acid bacteria both could stimulate the body to produce mucosal immunity,humoral immunity and cellular immunity against PEDV.Oral immunization of piglets withΔupp ATCC 393-S1 can also stimulate the body to produce mucosal immunity,humoral immunity and cellular immunity against PEDV,indicting the protective effect on PEDV infection.The results of this study have certain reference significance for the development of porcine PEDV oral vaccine. |
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