Immunoprotective Effects Offered By Recombinant Enterococcus Faecalis Expressing Key Domains Of Eimeria Tenella Microneme Protein 3 Against Homologous Infection

Chicken coccidiosis is caused by Eimeria infection,which brings huge economic losses to the poultry industry.The prevention and control measures of chicken coccidiosis mainly rely on drugs and/or live vaccines.However,due to potential hazards such as drug resistance,drug residues,and virulence revertion of live vaccines,the development of new vaccines to prevent coccidiosis has gradually become a research hotspot.Applying lactic acid bacteria as a vaccine carrier has the characteristics of easy culture,probiotics and immune adjuvant.With the development of bacterial surface display systems,the anchored expression of foreign proteins on the bacterial surface has been achieved.Studies have shown that Eimeria tenella（E.tenella）microneme protein 3（microneme protein 3,EtMIC3）has good immunogenicity.EtMIC3 has 7 tandem repeat structures,including A,B,C1,C2,C3,C4 and D,of which A,B and D are three incompletely conserved repeat structures located at both ends of the protein;C1,C2,C3 and C4 are located in the middle of the protein as fully conserved regions.In this study,MDXEF-1,the isolate of Entericoccus faecalis（E.faecalis）identified and preserved in our laboratory,was used as the host bacteria to express the BC1and C4D domains of EtMIC3 protein in a surface-displayed manner,and on the basis of surface anchoring,dendritic cell targeting peptide（DCpep）is introduced to enhance the uptake and presentation of dendritic cells,aiming to produce a stronger immune responses and protection against homologous infection.First,the recombinant E.coli expression strain BL21/p GEX-C4D was constructed.After induction with IPTG,SDS-PAGE was used to analyze the protein.The GST-tagged protein purification kit was used to purify the protein.The GST fusion protein C4D was immunized with New Zealand white rabbits to prepare polyclonal antibodies.The titer of rabbit antiserum was detected by ELISA and Western blot was used to test the specificity of the prepared antibody by reacting with the expressed BC1 protein in BL21/p ET30a-BC1（BC1and C4D protein have the same domain C）.The results showed that the serum titer of the prepared polyclonal antibody reached 2¹⁶,and the GST-C4D protein was expressed in BL21with a size of 53 k Da.A specific band of 41 k Da in size was detected by Western blot using BC1 protein,which has the same repeat domain C with C4D protein.The amplified C4D and BC1 fragments were cloned into the lactic acid bacteria expression vector p TX8048-SP-CWA and dendritic cell targeting peptide（DCpep）was introduced to obtain recombinant positive plasmids p TX8048-SP-BC1-CWA,p TX8048-SP-DC-BC1-CWA,p TX8048-SP-C4D-CWA and p TX8048-SP-DC-C4D-CWA,respectively.The four positive plasmids were electro-transformed into MDXEF-1 host bacteria to obtain four strains of recombinant E.faecalis,abbreviated as MDXEF-1/BC1-CWA,MDXEF-1/DC-BC1-CWA,MDXEF-1/C4D-CWA,MDXEF-1/DC-C4D-CWA.The four recombinant strains were induced by Nisin,and the above-mentioned rabbit-derived C4D polyclonal antibody or the rabbit-derived BC1polyclonal antibody preserved by our research group was respectively used to detect the expression of exogenous protein on the bacterial surface by using Western blot and indirect immunofluorescence.The results showed that BC1 or C4D protein of fusion or non-fusion DCpep can be expressed on the surface of recombinant E.faecalis.Afterwards,Nisin was used to induce the above four strains of recombinant E.faecalis and the empty plasmid control E.faecalis MDXEF-1,and the bacterial concentration was adjusted to 1.0×10¹¹CFU/m L,and 7-day-old chicks were immunized.Each chicken was orally immunized once a day,for three consecutive days,each time with 100μL(1.0×10¹⁰ CFU)bacterial solution,three times in total,with a 2-week interval between each time,The control and challenge control groups were orally administered 100μL of PBS（p H7.2）.Chicken serum,cecal lavage fluid and spleens were collected from each group at 14 days after each immunization.Indirect ELISA was used to detect levels of IgG in serum and s Ig A in cecal lavage fluid.The m RNA expression levels of cytokines IL-2,IFN-γ,IL-4,IL-10,IL-6 and IL-17 in spleen were detected by real-time quantitative PCR（q RT-PCR）.At two weeks after the third immunization,the peripheral blood of chickens in each group was taken to prepare lymphocyte suspension,and the proliferation of lymphocytes was detected by CCK8 kit.The results showed that the four recombinant strain groups could induce significantly increased specific serum IgG and mucosal s Ig A,as well as the expression levels of spleen cytokines IL-2,IFN-γ,IL-4,IL-10,IL-6 and IL-17,which gradually increased with the increase of immunization times.The antibody levels of evoked by the recombinant bacteria expressing DCpep was significantly higher than that of non-DCpep fusing bacteria（p<0.05）,and the level of immune responses induced by recombinant E.faecalis expressing BC1 protein was significantly higher than that of E.faecalis expressing C4D protein（p<0.05）.After three immunizations,the proliferation of peripheral blood lymphocytes in the four immunized groups with recombinant bacteria was significantly higher than that in the PBS and MDXEF-1 group,and the proliferation DCpep-fusing recombinant bacteria group was significantly higher than that of the non-DCpep-fusing bacteria group（p<0.05）.E.tenella infection experiment was carried out one week after the third immunization to evaluate the immune protection effects.Except for the PBS group,chickens in each group were fed with 4.0×10⁴ E.tenella sporulated oocysts.At 7 days after infection,the effects of immune protection were evaluated by analyzing weight gain,oocyst excretion rate,lesion score,anticoccidial index,observation of histopathological changes and the expression levels of inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-αin the cecal tissue.The results showed that,compared with the control group,the four of recombinant bacteria had higher weight gain rate,higher oocyst reduction rate,and less lesions,and MDXEF-1/DC-BC1-CWA group had the best effects;The anticoccidial index（ACI）of the four immunized groups,MDXEF-1/C4D-CWA,MDXEF-1/DC-C4D-CWA,MDXEF-1/BC1-CWA,and MDXEF-1/DC-BC1-CWA,were 161.69,172.68,171.47 and 178.41,respectively;The pathological and histopathological changes in cecum in each group after challenging showed that the lesions in the four recombinant bacteria groups were more mild than that of the control group,and the lesions in MDXEF-1/DC-BC1-CWA group were the least.The expression levels of inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-αof the four recombinant bacteria groups were significantly lower than that in infection control group（p<0.01）.The levels of each factor in the recombinant bacteria groups expressing DCpep were significantly lower than that in non-DCpep fusing groups（p<0.01）,and the bacteria group expressing BC1 protein were significantly lower than that in the bacteria group expressing C4D protein（p<0.01）.In conclusion,oral immunization with surface-displaying BC1 or C4D domain of EtMIC3 protein fused or non-fused DCpep E.faecalis can stimulate chickens to produce a specific immune response,provide partial immune protection against homologous infection.This study provide a reference for the study of lactic acid bacteria vaccine based on EtMIC3protein.