Study On The Immune Effect Of H7 Subtype Avian Influenza DGL Nano-vaccine

Avian influenza(AI)is a highly contagious disease caused by the avian influenza virus(AIV).The H7 subtype AIV is a highly pathogenic avian influenza virus(HPAIV),which is characterized by a short incubation period,acute onset,spread fast,high morbidity and mortality.Not only does it cause huge economic losses to the livestock industry,it also threatens human health.At present,effective methods for preventing and treating AI epidemic diseases are vaccination.But DNA vaccines have low immune efficiency,large doses,weak immunogenicity,multiple immunizations,and plasmid DNA degrades rapidly in vivo,resulting in low bioavailability.The protein expression level is low and the immune effect is not ideal.As a vaccine adjuvant and delivery vehicle,bio-nanomaterials can achieve sustained release of antigens,improve antigen presentation efficiency,enhance immunogenicity,avoid antigen degradation,induce mucosal immunity,and have become a hot topic in the field of nano vaccines and nanomedicines.This paper explored the prokaryotic expression of HA protein and NA protein of H7 subtype avian influenza virus,and prepared a polyclonal antibody of the protein,which laid a solid theoretical foundation for the study of the protein function of the H7 subtype avian influenza virus and the subsequent experimental gene expression and identification.At the same time,the H7 subtype avian influenza virus HA gene and different NA gene fragments were inserted into the eukaryotic expression vector Huang4 to construct 9 eukaryotic expression plasmids.In addition,the plasmid with the best expression of the protein screened by the animal in vivo immunoassay was used as a model antigen,and the nanomaterial DGL as the gene delivery vector,the nano vaccine(pβH7N2SH9/DGL NPs)was prepared that can simultaneously express the HA protein of H7 subtype AIV and the related NA protein,and the pβH7N2SH9/DGL NPs of physicochemical properties,anti-DNase I degradation,in vitro release,and evaluation of immune effects were studied.The results showed as follow:1.Successfully optimized avian influenza gene fragments,and constructed 9 eukaryotic expression plasmids containing H7 subtype avian influenza virus HA gene and different avian influenza NA gene;2.The prokaryotic expression of HA protein and related NA protein of H7 subtype avian influenza virus showed that the polyclonal antibody was successfully prepared and the eukaryotic expressed plasmid target gene fragment protein could be expressed;3.The results of animal immunoassay showed that the plasmid pβH7N2SH9 containing 3 target gene fragments was superior to the plasmid containing one or two gene fragments of interest.The effect of plasmid pβH7N2SH9 immunized mice was the best.;4.pβH7N2SH9/DGL NPs was successfully prepared.In vitro release showed that the release of plasmid DNA in the nano vaccine was first released and reached a certain level after sustained release slowly;5.DGL cytotoxicity test results show that: DGL is less toxic,safe and efficient,can be used as plasmid pβH7N2SH9 delivery vector;6.The prepared pβH7N2SH9/DGL NPs were characterized by regularity,uniform size and good dispersibility.The average particle size was(68.38±1.89)nm(n=3)and the zeta potential was(15.40±1.41)mV(n=3).The rate was(91.25±1.83)%(n=3),and the drug loading was(30.52±0.78)%(n=3).7.In vitro DNase I digestion test showed that DGL can protect pβH7N2SH9 from DNase I degradation;8.The results of animal immunization showed that the overall titer of antibody titer in the intramuscular injection of pβH7N2SH9/DGL NPs immunized group was significantly higher than that of plasmid pβH7N2SH9 immunized group(p<0.05),and there was no significant difference among groups(p>0.05).No antibody was produced in the PBS and the DGL immunized group at all times;indicating that the humoral immunity level of the body was significantly increased after the DGL encapsulated the plasmid pβH7N2SH9;9.Cell-level assays showed that the secretion of IL-2 and IFN-α in the immunization group with pβH7N2SH9/DGL NPs was significantly higher than that in the plasmid pβH7N2SH9 group(p<0.05),and the difference between IL-2 and DGL and PBSimmunized group was extremely significant(p<0.01).CD4+/ CD8+ and CD3+ CD4+ T lymphocyte number increased rapidly and induced a cellular immune response.In this study,a plasmid expressing the HA protein of H7 subtype avian influenza virus and related NA protein was constructed,and a H7 subtype avian influenza DGL nano vaccine was prepared to improve its transmembrane transport and expression,providing a reference for H7 subtype AIV prevention and the promotion of veterinary vaccine replacement.The development of new vaccines provides research ideas and methods that can be used for reference.It has certain academic research value and potential application prospects.