Preparation And Immune Effect Evaluation Of HA Protein Nanoparticle Vaccine Of H5 Subtype Avian Influenza Virus

The wide spread of H5 subtype Avian influenza Virus（AIV）in the world poses a great threat to the breeding industry and public health security.Vaccination is the most cost-effective way to prevent avian influenza virus infection.The traditional inactivated chicken embryo avian influenza vaccine has played an important role in the prevention and control of avian influenza,but the inactivated chicken embryo vaccine mainly induces humoral immunity and requires multiple vaccinations to achieve better protection,which brings a lot of inconvenience to the actual production.Subunit vaccine can induce humoral and cellular immune responses simultaneously,and a single inoculation can obtain longer duration of immune protection.Due to the bionic characteristics of pathogens,nanoparticle subunit vaccines have shown unique advantages in antigen assembly and antigen presentation,and have shown broad application prospects in the prevention and treatment of major diseases such as COVID-19,influenza,hepatitis B and tumor in recent years.Influenza virus hemagglutinin（HA）protein,as the main protective antigen,can induce a high intensity protective immune response in the body.A tetravalent influenza vaccine targeting HA has completed p Hase III clinical trial.Therefore,this study took H5 subtype avian influenza as the research object to develop a safe and efficient influenza HA protein nanoparticle vaccine.Specific tests are as follows:1.The hemagglutinin（HA）protein was screened as a candidate antigen according to the sequence of 2021 H5 subtype avian influenza virus epidemic strain.The recombinant plasmid p BACPAK9-HA was successfully constructed by fusing the HA gene into the p Bac PAK9vector framework by homologous recombination.2.The recombinant plasmid and linearized Bacmid were transfected into Sf9 cells through lipid transfection,and the recombinant HA protein was obtained by High Five expression.The expression products of High Five cells were collected for 96 h and lysed with lysate containing Triton 100.The lysate supernatant was filtered by 0.45μm filter membrane and purified by Q column and lectin affinity column.The hydrop Hobic region of the stem of HA and the core of PS80 detergent bonded on the hydrop Hobic affinity column to form nanoparticles,which were eluted by high concentration of PS80 buffer to obtain high purity ha-PS80 nanoparticles protein.SDS-PAGE and gray scan showed that the purity of HA protein was more than 85%.Western blot analysis showed that the recombinant HA protein could react well with mouse polyclonal serum of H5 subtype.Transmission electron microscopy showed that the head region of HA protein protruded outward,and the stem hydrop Hobic region and PS80 detergent formed an antigenic sp Herical core,which could bind2～7 HA trimers per nanoparticle core.DLS dynamic light scattering detection showed that the size of nanoparticles ranged from 25 nm～35 nm.HA nanoparticle protein of H5 subtype AIV was successfully prepared.The HA nanoparticle protein showed good stability at 25℃.3.The purified HA nanoparticle protein was mixed with ISA 206 adjuvant to prepare vaccine and immunized SPF chickens.SPF chickens were injected subcutaneously with 5μg/300μL/chick（low dose group）,15μg/300μL/chick（medium dose group）,30μg/300μL/chick（high dose group）,300μL/chick commercial vaccine and 300μL/chick PBS negative control group,respectively.0d,7d,14d and 21d,28d and 35d,42d blood,separation of serum and blood clotting inhibition experiment was carried out.The results showed that the antibody titer of the high dose group was up to 1:1024 at 28 days after immunization,which was more than 4 times that of the commercial vaccine with the same dose.The SPF chickens were challenged with 10^6.57TCID₅₀ H5N8 virus at 28 days after immunization.The results showed that no obvious clinical symptoms and death occurred in the MEDIUM and high dose immunization groups at 72 hours after immunization,and the protection rate was 100%（10/10）.Low dose group showed mild clinical symptoms,some chickens died,and the protection rate of challenge was 80%（8/10）.The results showed that SPF chickens could be protected against H5 subtype AVIAN influenza virus infection by 15μg/300μL of AIV H5subtype HA nanoparticle protein vaccine.In conclusion,in this study,the recombinant plasmid p BACPAK9-HA of AIV H5 subtype HA gene was successfully constructed and expressed by insect baculovirus expression system.After purification by ion column and affinity column,high purity HA nanoparticle protein was obtained.SPF chickens were immunized with vaccine.Provides adequate protection against H5 subtype avian influenza virus infection.This study provides a basis for the development of H5 subtype avian influenza nanoparticle vaccine.