Preparation Of LDH@SiO₂ Shell-core Nanoparticles Carrying HA Gene Of H9N2 Avian Influenza Virus And Its Immune Effect

Avian influenza（AI）is a disease caused by poultry infected with avian influenza virus（AIV）.Among many preventive measures,vaccination is one of the best ways.The DNA vaccine is a new generation of vaccine in the contemporary society,but the naked plasmid is easily degraded by the nuclease in the delivery process,and the application is limited by the disadvantages of low utilization rate.At present,nanomaterials have been widely studied and studied by the whole society as a vector of plasmid DNA.The use of nanomaterials as a carrier can effectively complement the disadvantages of DNA vaccine,which can protect DNA from degradation,increase transfection rate and enhance immune effect.In this experiment,silicon dioxide（SiO₂）and magnesium aluminum double hydroxide（LDH）were co-precipitated to synthesize shell-core nanocomposites:LDH@SiO₂.The plasmid pβH9N1SH5 expressing HA protein of H9N2 subtype AIV was used as a model drug to prepare pβH9N1SH5/LDH@SiO₂ NPs,and its physicochemical analysis,stability test,transfection test,and immunity test were carried out.The evaluation of the epidemic effect.The results of the test are as follows:1)The prepared LDH has a regular morphology,a flaky structure,good dispersibility,an average particle size of（925.60±21.91）nm,the dispersion coefficient is 0.354,a Zeta potential of（45.30±0.63）mV;2)The LDH@SiO₂ shell-nuclear nanocomposite material was successfully synthesized.The appearance of LDH@SiO₂ was complete,and an average particle size of（357.31±2.03）nm,the dispersion coefficient is 0.005,a Zeta potential of（29.81±1.06）mV;3)The prepared pβH9N1SH5/LDH@SiO₂ NPs were intact,the entrapment efficiency was（85.21±0.89）%（n=3）,and the drug loading was（47.32±1.45）%（n=3）.4)The in vitro release test showed that the release of DNA was first released and released slowly after in vitro conditions of pβH9N1SH5/LDH@SiO₂ NPs;5)The results of DNA protection assay assay showed that LDH@SiO₂ can protect pβH9N1SH5 from nuclease degradation;6)In vitro expression assays showed that pβH9N1SH5/LDH@SiO₂ NPs could be successfully transfected into 293T cells and express the protein;7)CCK-8 toxicity test showed that pβH9N1SH5/LDH@SiO₂ NPs were less toxic and the safety factor was higher;8)Mouse immunoassay showed that intramuscular injection of pβH9N1SH5/LDH@SiO₂ NPs could stimulate the mice to produce IgG antibodies,and the antibody level was higher than other groups;the result of ELISA for IFN-γand IL-2 was significantly higher in the spleen lymphocyte proliferation test than in the other groups.In this experiment,nanomaterials with the ability to carry macromolecules loaded with SiO₂ and the advantages of LDH layered structures were successfully prepared.Avian influenza DNA vaccines with LDH@SiO₂ as a carrier effectively protected DNA and achieved long-term effects.It has a certain application prospect.