The Preparation Of Avian Influenza DNA Vaccine With DGL As Carrier And Its Immune Mechanism

Avian influenza(AI)is an infectious disease caused by avian influenza virus(AIV).Vaccination is one of the main methods to prevent AI.DNA vaccine is a kind of promising vaccine due to its characteristics of facile construction and production,easy storage and distribution.However,plasmid DNA is easy to be degraded by the nucleic acid enzymes in the body when it is inoculated directly by intramuscular injection,which leads to the decrease of the biological utilization rate and results in large dosage,weak immunogenicity and low protein expression level.Now,it has attracted wide attention that the biodegradable nanomaterials used as carriers could protect DNA from the damage by the body enzymes,promote absorption by cells,and enhance the immune response in hosts.In this study,the eukaryotic plasmid(p CAGGS-opti441-HA)expressing HA protein was constructed by inserted optimized HA gene of H9N2 AIV into p CAGGS vector firstly.Then the p CAGGS-opti441-HA/DGL was developed through packaging p CAGGS-opti441-HA by polylysine dendrimers DGL by electrostatic adsorption.The physicochemical characters,stability,transfection efficiency,immune response of the p CAGGS-opti441-HA/DGL were studied and evaluated.The results demonstrated that:1.The cytotoxicity assay of DGL showed that it was safe,non-toxic and suitable for carrying DNA in vaccine development.2.The p CAGGS-opti441-HA/DGL had been produced with a regular morphous.The particle size of the p CAGGS-opti441-HA/DGL was 68.88±2.07 nm and zeta potential was 55.13±1.32 m V.3.DNase?digestion test showed that DGL had the ability to protect p CAGGS-opti441-HA from DNase?degradation.4.In vitro expressing test suggested that the process of preparation of p CAGGS-opti441-HA/DGL didn't destroy the biological activity of plasmid DNA.5.The release test of p CAGGS-opti441-HA/DGL in vitro demonstrated that DNA was released continuously after initial burst release.6.Animal vaccination test showed that humoral immune respons in specific pathogen-free(SPF)chickens inoculated intramuscularly with the p CAGGS-opti441-HA/DGL was induced and antibody levels were higher than those of other immune groups.The high antibody levels maintained for more than 10 weeks after a single immunization.7.Strong cellular immune responses could be induced in the SPF chicken after inoculated intramuscularlu with p CAGGS-opti441-HA/DGL.The concentrations of IFN-? and IL-2 in serum,and lymphocyte transformation rate of vaccinated chickens increased at the third weeks post initial immunization.In the vaccinated SPF chickens,the T lymphocytes in peripheral blood were activated and proliferated,and the number of CD3+CD4+ and CD4+/CD8+ T lymphocytes increased rapidly,indicating that p CAGGS-opti441-HA/DGL induce a certain level of cellular immune response.8.The SPF chicken was challenged by inoculating H9N2 AIV intranasally or introvenously.The SPF chickens vaccinated intramuscularly with p CAGGS-opti441-HA/DGL were protected completely against the H9N2 AIV challenged by introvenous injection.Though the SPF chickens vaccinated intramuscularly with p CAGGS-opti441-HA/DGL were not protected completely against the H9N2 AIV challenged by intronassally inoculation,the virus titers in the swabs of decreased significantly when comparing with other groups.In this study,the p CAGGS-opti441-HA/DGL vaccine was prepared based on DGL nano delivery system which enhanced the efficiency of cell membrane transport and expression of DNA vaccine,improved the level of humoral and cellular immune response.Our study provides not only a new method for the development of novel AIV vaccines,but also a theoretical basis for the development of safe and efficient gene delivery carriers.