Construction Of Lactobacillus Casei Expressing PEDV Protective Antigen Targeted Fusion Peptide And Immunogenicity Analysis

Porcine epidemic diarrhea(PED) is a kind of intestinal infectious diseases caused by porcine epidemic diarrhea virus(PEDV), and its clinical features include severe diarrhea, vomiting and dehydration. PED can occur in pigs of all ages, but piglets are the most susceptible, and the mortality rate can reach 95%. PEDV infection has marked intestinal tropism, so mucosal immunization can effectively prevent the occurrence of the disease.To achieve good intestinal mucosal immune response, we chose ps420 fragment of PEDV S protein as target antigen and directionally inserted into lactic acid bacteria expression vector, and then electrotransformed into lactic acid bacteria, which was used as oral vaccine to explore its specific immune effect. To improve antigen uptake and presentation efficiency, M cell target peptide gene sequence co1 and dendritic cells target peptide gene sequence DCpep were introduced to enhance immune response.The clinical suspected PED specimen was used to amplify ps420 frament. Then ps420 was cloned into p ET-32 a and transformed in E.coli BL21(DE3). Determine the expression of the target protein by SDS-PAGE and Western blot, and then purify the protein and use for the detection of splenic lymphocyte proliferation.Introduce M cell targeting peptide co1 gene and dendritic cell targeting peptide DCpep gene by SOE-PCR, and we got genes ps420, ps420-co1, ps420-DCpep and ps420-co1-DCpep. Then the genes were inserted into the Lactobacillus expression vector pPG-T7g10-PPT and transformed into Lactobacillus casei 393, and we obtained four positive recombinant bacteria, named non targeted p PG-T7g10-PPT-ps420/L. casei 393, single M cell targeting p PG-T7g10-PPT-ps420-co1/L. casei 393, single dendritic cells targeting p PG-T7g10-PPT-ps420-DCpep/L. casei 393 and dual targeting M cells and dendritic cells p PG-T7g10-PPT-ps420-co1-DCpep/L. casei 393.The four recombinant Lactobacillus were cultured respectively at 37℃ for about 16 hours, and detected by Western blot, the results showed that recombinant strains expressed target protein in the expected position. The results of laser scanning confocal microscope showed that the four strains of recombinant bacterial expressing ps420 protein showed visible yellow green fluorescence, but the control bacteria pPG-T7g10-PPT/L. casei 393 didn’t, showing that the recombinant Lactobacillus expressed the target protein and was displayed on the cell surface.In order to investigate the effect of recombinant Lactobacillus on immune response level, this study used BALB/c mice as animal model. Different groups were oral immuned different recombinant Lactobacillus, and PBS and p PG-T7g10-PPT/L. casei 393 were used as control. Programs were divided into two: once immunization group and twice immunization group, inoculation dose was 100 μL, containing live bacteria number 1×109 CFU. Collect the fresh feces, blood of mice, genital mucus samples, and intestinal mucus, and detect the antibody levels and cytokine levels with different methods: ELISA, MTT and splenic lymphocyte proliferation kit.The results of ELISA showed that pPG-T7g10-PPT-ps420-co1/L. casei 393, p PG-T7g10-PPT-ps420-DCpep/L. casei 393 and p PG-T7g10-PPT-ps420-co1-DCpep/L. casei 393 immunized mice can obtain high level IgG and s IgA antibody on day 7 after immunization, which were significantly higher than non-target group pPG-T7g10-PPT-ps420/L. casei 393(p<0.05) and control group(p<0.01), and the immune effect of dual targeting modification recombinant L. casei 393 was higher than that of single targeted modification recombinant Lactobacillus. After twice immunization, the antibody level increased significantly, which was statistically significant difference between groups(p<0.05) and significantly higher than with the control group(p<0.01). The experimental results showed that serum Ig G antibody possessed virus-neutralization activity. In addition, the detection of spleen lymphocyte proliferation and cytokine levels showed that recombinant lactic acid bacteria can induce cellular immune response. These results indicate d that the recombinant Lactobacillus casei expression system can stimulate mucosal immune responses and systemic immune response, while the target-modification groups showed better immunogenicity and the dual target-modification recombinant strain possessed the best immunogenic.In summary, this study constructed recombinant Lactobacillus expression system which express PEDV protective antigen, and prove d that M cells and dendritic cells-double target strategy is indeed feasible in lactic acid bacteria live vector system. The expression systems possess potential application value and lay foundation for the further development of new type PEDV oral vaccine.