| The H9N2 subtype influenza is a type of low pathogenic infectious disease caused by Orthomyxoviridae and Influenza A viruses.The pathogen is a single-stranded negative-strand RNA virus and is divided into 8 segments.Viral particles are divided into three layers,the inner layer is a nuclear shell;the middle layer is the matrix;the outer layer is a capsule,and the surface is interspersed with haemagglutinin(HA)that can agglutinate more than 20 red blood cells(mammalian and avian)and neuraminidase(NA),HA is the main antigenic determinant of influenza virus.,can directly induce the production of virus-neutralizing antibodies.However,antigenic drift often occurs in the amino acid sequence in HA,and the recombination between different segments of the RNA gene can also lead to the generation of novel HA molecules(antigen conversion)by the influenza virus strain,which requires constant updating of the vaccine in prevention and control the avian influenza.In the vaccines used to prevent and control bird flu,the inactivated or attenuated vaccine itself mainly induces a humoral immune response,and there is a risk of viral spread,virulence return or residual virulence,and the mutual interference between different vaccines is easily generated.Compared with traditional vaccines,DNA vaccine as a novel vaccine has the advantages of long-lasting response,safety and stability,and easy preparation and storage,and can cause stronger CTL responses in cellular immunity,and can be used to construct a multi-gene fusion expression plasmid to simultaneously achieve resistance to invasion of multiple pathogens.The aim of this experiment is to enhance the immune response of H9N2 subtype HA-DNA vaccine to SPF chickens.The C3 d complement component which had the immunomodulatory activity was used as the study object,and its specific receptor binding region P29 was used as a molecular adjuvant.The HA gene and different copy number of C3d-P29 were cloned into the pVAX1 expression vector in order to construct pVAX1-HA and pVAX1-HA-P29.n(1,2,4)fusion expression plasmids.The above plasmids were transfected into 293 T cells by liposome transfection method for in vitro expression experiment,and IFA detection of the transfected cells demonstrated its specific fluorescence.The constructedpVAX1-HA,pVAX1-HA-P29.n(1,2,4)and control pVAX1 plasmids were immunized at a dose of 50 μg/feather of SPF chickens at 3 weeks of age by multiple intramuscular injections,after 14 days,strengthen immunity with the same dose,serum collected weekly after the first immunization.The levels of serum cytokines IFA-γ and IL-4 and anti-HA(H9N2)antibody titer were detected by indirect ELISA.The results showed that the levels of IL-4,IFA-γ,and anti-HA antibodies in the fusion-expressed HA-P29.n(1,2,4)groups after immunization were significantly higher than those in the pVAX1-HA group,and there is a certain positive correlation between fusion P29 concatenated numbers and the contents or antibody levels.The study found that C3d-P29 promoted the early generation of IFN-γ,the greatest difference of IL-4 levels between the groups occurred after 14 days of second immunization,and the content of IL-4 in the pVAX1-HA-P29.4 group was the highest,indicating that tandem P29 fragments promote the secretion of IL-4.The proliferation of chicken spleen lymphocytes was detected by MTT assay,the result shows that the specific lymphocyte proliferation induced by pVAX1-HA-P29.n(2,4)group was significantly higher than that of pVAX1-HA-P29.1 and pVAX1-HA groups.The amount of detoxification detected by qPCR after challenge,histopathological observation and detection of serum HI antibody also showed that the pVAX1-HA-P29.n(2,4)group had better protection after immunization.This study was conducted to study the effect of the fusion plasmids which included avian complement C3 d fragment multiple tandem receptor binding region P29 and the H9N2 subtype AIV-HA gene on the immune response of SPF chickens.The results showed that the C3d-P29 complement fragment as a molecular adjuvant can enhance the specific humoral and cellular immune responses induced by AIV-HA gene,laying the foundation for further study of the optimal conditions of C3d-P29 acting as a molecular adjuvant on AIV nucleic acid vaccine. |
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