Immune Evaluation Of Attenuated Salmonella Expressing 3M2e-Ferritin-DCpep Against H9N2 Subtype Avian Influenza Virus

H9N2 subtype avian influenza virus(Avian Influenza Virus,AIV)was first isolated in China in 1994 and has gradually become popular,which has had a serious impact on China’s poultry industry.During infection,chickens usually have no clinical symptoms,but can increase the incidence of infection with other respiratory pathogens.Morbidity during coinfection.An effective measure to prevent influenza virus infection is vaccination.Most currently available inactivated influenza vaccines include two major viral surface proteins,Neuraminidase(NA)and Hemagglutinin(HA),which induce Antibodies react to prevent antigen matching.Because HA and NA antibody responses are largely subtype-specific,conventional influenza vaccines do not provide effective allotype immunity.The research on universal vaccines targeting the conservative antigens of influenza virus has aroused the interest of researchers in recent years.A variety of conservative antigens including M2e can theoretically be used as candidate antigens for universal vaccines,but their own immunogenicity is low.Improving its immunogenicity is a scientific problem that researchers hope to solve.In this study,the self-assembly and modifiable properties of ferritin nanoparticles were used to fuse and express a copy of the influenza virus conserved antigen M2e3(3M2e)and dendritic cell targeting peptide(DCpep)with the N-terminus of ferritin monomer.Self-assembled into 24-mer nanoparticles and displayed 3M2e on the surface of the nanoparticles,constructed a recombinant strain of 3M2e-Ferritin self-assembled nanoparticles,named pYL180N;and then constructed 3M2e-Ferritin-DCpep dendritic cell-targeted nanoparticle The particle expression vector was named pYL240N;in addition,a recombinant bacterium expressing 3M2e was constructed and named pYL179N as a control to compare the immunogenicity and immunoprotective effect of the three.First,the expression of the target protein and the successful assembly of nanoparticles were verified by Western-blot and transmission electron microscopy,and then 1-day-old chicks were immunized with recombinant bacterial solution orally.One week after the fourth immunization,Ig Y antibody in the serum of the chicks was detected by ELISA,and tracheal lavage was performed.s Ig A antibody in lavage fluid,bronchoalveolar lavage fluid and intestinal mucosal lavage fluid;CCK-8 assay to detect spleen T lymphocyte proliferation;commercial ELISA kit to detect IFN-γcontent in cell supernatant;flow cytometry to detect spleen Differentiation of CD3~+CD4~+,CD3~+CD8~+in T lymphocytes.Two weeks after the fourth immunization,the H9N2 AIV A3 strain was used to challenge the virus,and the serum Ig Y antibody and mucosal s Ig A antibody were detected on the 5th day after the challenge;MDCK cells were used to detect the virus titer TCID50 in the lungs of chicks;Pathological tissue sections were observed to observe the pathological changes.In addition,this study performed 16SrDNA sequencing on the genomic DNA of cecal mucosal flora 5 days after infection with H9N2 subtype avian influenza A3 strain to analyze the effect of H9N2AIV on the intestinal flora of chicks and the effect of attenuated Salmonella immunization on the intestinal flora of chickens.group protection.The main contents and results of this study are as follows:(1)Construction and expression verification of 3M2e,3M2e-Ferritin,3M2e-Ferritin-DCpep recombinant strainsIn this study,the target fragments of 3M2e,3M2e-Ferritin,and 3M2e-Ferritin-DCpep were ligated into pYL152 expression vector,and pYL179N,pYL180N and pYL240N were successfully constructed.The expression of the target protein was verified by Western-blot,and self-assembled nanoparticles with a diameter of about 15 nm were observed by transmission electron microscopy.(2)Evaluation of the immune effect of recombinant strainsThe plasmids pYL179N,pYL180N and pYL240N were electroporated intoχ11802 in Salmonella to obtain recombinant strainsχ11802(pYL179N),χ11802(pYL180N)andχ11802(pYL240N).Female laying hens were selected as experimental animals,and broiler chickens were immunized on the 0th,14th,28th,and 42nd days(the first immunization was 0 days),and the immunization dose was 1×10~9CFU/feather.In this experiment,the production of Ig Y in serum and s Ig A antibody in mucosa was detected by ELISA after immunization.The results showed that the immunized strains produced more obvious s Ig A antibody in mucosa,and the pYL240N group had the best effect.According to the detection results of spleen T lymphocyte activation ability,χ11802(pYL240N)andχ11802(pYL180N)can more effectively stimulate lymphocyte proliferation,and flow cytometry results showed that the ratio of CD4~+/CD8~+T cells in theχ11802(pYL240N)immune group was higher high.In addition,theχ11802(pYL240N)immunized group had the highest s Ig A antibody content in the alveolar lavage fluid 5 days after challenge.The results of lung pathological section and lung virus load test also showed that theχ11802(pYL240N)immunization group had the best effect.(3)16SrDNA sequencing of cecal mucosal floraH9N2 subtype avian influenza virus infection caused changes in the intestinal flora of chicks and inhibited the growth of probiotics.After immunization,the number of probiotics in the intestines of chicks in the pYL240N group increased and could return to the original normal flora structure.The results of this study show that the recombinant strain of pYL240N self-assembled nanoparticles has a certain protective effect against H9N2 subtype avian influenza,which lays a certain preliminary foundation for the study of universal influenza vaccine.