Protective Effects Of Lactic Acid Bacteria Expressing Dendritic Cell Peptide Fusion With Chicken Eimeria Tenella 3-1E Protein

Chicken coccidiosis is caused by Eimeria,which is a major infestation of a parasitic disease of chicken intestine.The disease severely restricts the development of large-scale breeding,and brings serious economic losses to the poultry industry.At present,the control of chicken coccidiosis is mainly through the prevention of drugs,but due to a series of reasons such as drug resistance,drug residues,and economic costs,we must seek more reasonable and effective preventive measures.As a food-grade microorganism,lactic acid bacteria has been gradually developed into a carrier for oral vaccines in recent years.In the previous study,we expressed the Eimeria sporozoites invasion-associated protein 3-1E on the surface of lactic acid bacteria.Animal experiments showed that the recombinant lactic acid bacteria expressing 3-1E protein produced immunity against coccidiosis after immunization of chickens.Based on this research,the surface expression system was modified,the DC targeting peptides（DCpep）were fused with the 3-1E encoding gene on the surface of the coccidia.The recognition and uptake of target antigens by dendritic cells results in a stronger specific immune response.The animal immune experiments were used to test the level of immune response induced by the constructed recombinant lactic acid bacteria,and the effect of anticoccidial immunity after immunization with lactic acid bacteria was evaluated.1.Construction of recombinant lactic acid bacteria that express DCpep and 3-1E protein.The synthesized fragment containing the L.lactis NZ9000 Usp45 signal peptide and DCpep targeting peptide（SP-DCpep）was cloned into the lactic acid bacteria surface expression plasmid pTX8048-SP-3-1E-CWA instead of the SP fragment.The correct positive plasmid was electrotransformed into L.lactis NZ9000 and E.faecalis MDXEF-1（Enterococcus faecalis）and positive recombinant lactic acid bacteria were screened.L.lactis NZ9000/pTX8048-SP-DCpep-3-1E-CWA and E.faecalis MDXEF-1/pTX8048-SP-DCpep-3-1E-CWA,two kinds of positive bacteria were induced with Nisin at a final concentration of 5 ng/mL,and the expression of target protein was verified by Western-blot.The results showed that the target protein was successfully expressed in L.lactis NZ9000 and E.faecalis MDXEF-1 with a size of 53 kDa.2.Oral immunization of SPF chickens with recombinant bacteria.The recombinant strains were induced with Nisin（5 ng/mL）for 3 h and then centrifuged at 8000 rpm（4℃）for 12 min.The bacterial pellets were washed three times with sterile water and then suspended with a certain amount of PBS.E.faecalis MDXEF-1 recombinant strains concentration was adjusted to 5×10¹⁰CFU/mL,and the L.lactis NZ9000 recombinant strain was adjusted to 1×10¹11 CFU mL.A total of300 SPF chickens were divided into 8 groups with 35 animals in each group.All groups of chickens were immunized at 5-7,21-23,and 37-40 days of age.100μL of recombinant lactic acid bacteria were orally administered to each chicken.100μL of PBS was administered orally to the control group and the infection control group.On the 14th day after each immunization,blood samples were taken from the hearts of five chickens.Preparation of chicken cecal fluid for each group.Serum IgG and cecal fluid IgA antibody levels were measured by indirect ELISA;On the10th day after the third immunization,the peripheral blood of each group of chickens was used to prepare lymphocyte suspension.CCK8 kit was used to detect the proliferation of T lymphocytes in peripheral blood after the third immunization.Flow cytometry was used to detect the change of T lymphocyte subtypes in peripheral blood.After the third immunization,each group of chicken spleen were taken.The expression of IL-2 and IFN-γmRNA in the spleen were detected by Real-time fluorescent quantitative PCR.The results showed that with the progress of immunization,the serum IgG antibody and IgA antibody in the cecal fluid of the recombinant bacteriophage expressing 3-1E protein showed an upward trend,and the antibody level of the DCpep fusion bacterium immunized group increased compared to the non-fusion expression group.（P<0.05）;3.Evaluation of protective effect of anticoccidial infection after immunization with recombinant lactic acid bacteria.After the three immunizations,except for the PBS group,2×10⁴ E.tenella sporulated oocysts were orally administered to each group,and chicken in each group were all sacrificed after seven days later.The efficacy of immunoprotection was evaluated by testing indicators such as weight gain,cecal lesion score,reduced oocyst excretion rate,and anticoccidial index.The results showed that DCpep fusion expression group was superior to the non-fusion expressiongroup,E.faecalisMDXEF-1/pTX8048-SP-DCpep-3-1E-CWA,E.faecalis MDXEF-1/pTX8048-SP-3-1E-CWA,L.lactis NZ9000/pTX8048-SP-DCpep-3-1E-CWA,L.lactis NZ9000/pTX8048-SP-3-1E-CWA,E.faecalis MDXEF-1,L.lactis NZ9000 six groups the average weight gain was:106.75 g,99.03 g,111.28 g,106.61 g,54.81 g,and 50.43 g.The cecal average lesion scores were:1.6,2.4,1.4,1.8,3,3.6,and the reduction rate of oocysts was:38.52%,32.09%,41.75%,35.23%,11.38%,3.64%,anticoccidial index:166.21,155.57,171.75,163.96,112.73,107.68.The pathological changes and histopathological changes of the cecum of the four recombinant bacterial immunization groups were significantly reduced compared with the control group.The above results showed that DCpep and 3-1E protein can enhance the body’s cellular and humoral immune response levels.The four recombinant lactic acid bacteria in the experiment have a certain degree of immunoprotective effect on coccidiosis infection.Laid the foundation for a lactic acid bacteria-based dendritic cell-targeted oral vaccine.