Evaluation Of Immune Effect Of Salmonella Co-Expressing Mosaic HA Protein,M1 Protein Of H9N2 Subtype Avian Influenza

Avian influenza（AI）is a highly contagious disease caused by influenza virus.Among them,H9N2 subtype avian influenza is easily infected.Generally,vaccination is the most effective way to prevent influenza virus.However,at present,commercial vaccines rely on chicken embryos and tend to induce humoral immunity.Therefore,it is of great significance to study new vaccines against H9N2 subtype avian influenza virus.The main contents and results are as follows.1.Construction and Expression Verification of Microloop DNA VectorIn this study,the full-length Mosaic HA protein and M1 protein were connected to the micro-circle DNA（mc DNA）expression system constructed by our research group in the early stage through P2A sequence,and the micro-circle plasmid p YL221 containing double CMV promoters was successfully constructed.Then,the optimized adjuvant ch GM-CSF expression frame was added to the p YL221 plasmid,and the micro-circle plasmid p YL233 was successfully constructed.The expression of Mosaic HA protein,M1 protein and adjuvant ch GM-CSF were verified by indirect immunofluorescence and Western-Blot（WB）.2.Evaluation of immune efficacy of mc DNA vector for delivery of H9N2 subtype avian influenza with Salmonella as carrierIn this study,21-day-old laying hens were selected as experimental animals,and chickens were immunized for four times,with an immunization dose of 1×10⁹CFU/feather.The results of ELISA one week after immunization showed that the level of HA protein-specific s Ig A inχ11218（p YL221）andχ11218（p YL233）groups increased.One week after immunization,the results of ELISA showed that the specific Ig Y antibody levels of HA protein and M1 protein inχ11218（p YL233）group were significantly increased.The results of flow cytometry analysis showed that the number of CD3⁺CD4⁺T cells inχ11218（p YL221）group andχ11218（p YL233）group was significantly higher than that in BSG group（P<0.001）.The number of CD3⁺CD8⁺T cells inχ11218（p YL221）andχ11218（p YL233）groups was significantly higher than that inχ11218（p YA4545）empty vector group（P<0.05）.The results of lymphocyte proliferation test showed that compared with BSG group,χ11218（p YL233）group significantly enhanced the proliferation of T lymphocytes in spleen（P<0.05）.Quantitative PCR was used to detect IFN-γand IL-4 m RNA levels in spleen cells.The results showed that when HA protein stimulated spleen cells in vitro,IFN-γm RNA levels inχ11218（p YL221）group were significantly higher than those in BSG group（P<0.05）,while M1 protein stimulated spleen cells in vitro,χ11218（p YL221）group,χ11218（p YL221）Two weeks after immunization,chickens were challenged.The results of ELISA showed that compared with the control group,χ11218（p YL233）andχ11218（p YL221）groups had a certain increasing trend in the level of single protein-specific s Ig A antibody and Ig Y antibody.Using MDCK cells to detect the virus titer in chickens’lungs showed that it was empty withχ11218（p YA4545）.3.Effect of recombinant Salmonella on intestinal flora of H9N2 subtype avian influenza16Sr DNA sequencing analysis of chicken cecum showed that phylum and phylum level analysis showed that salmonellaχ11218（p YL221）andχ11218（p YL233）could increase the abundance of sclerenchyma,and the effect of p YL233 group on increasing the abundance of Bacteroides was better than that of p YL221 group.Cluster analysis of species abundance showed that Sclerotinia was still the dominant flora in intestinal flora after Salmonella immunization.Through diversity analysis,it is found that Salmonella immunization can significantly increase the richness and diversity of flora.This study indicated that delayed lysis recombinant Salmonella containing P_CMVdouble promoter co-expressing Mosaic HA protein and M1 protein of H9N2 subtype avian influenza had certain protective effect.It is worth pointing out that although ch GM-CSF adjuvant can enhance the body-specific immune response in some aspects,the effect of ch GM-CSF adjuvant in our recombinant bacteria as a whole has not reached the expected effect,and other more efficient adjuvant molecules will be selected for research in the future.