Preparation And Biological Activity Detection Of Newcastle Disease Virus F Gene LDH @ SiO <2> /

Newcastle disease (ND) is a highly contact resistance infectious disease of poultry caused by Newcastle disease virus (NDV), which causes serious threats to animal husbandry of the world that is the first-class national animal infectious diseases. Now the main method to prevent Newcastle disease is still the vaccine inoculation. The NDV inactivated vaccines and attenuated live vaccines are used universally to control ND. However, these conventional vaccines have some disadvantages in clinical application. DNA vaccine is superior than traditional vaccines, but it is easy to be degraded in vivo, low bioavailability and so on. However, using biodegradable materials in order to protect DNA from nuclease degradation, improve the transfection efficiency, reduce vaccination times and enhance the immune efficiency. So, in this study, the pFDNA-LDH@SiO2-NPs were obtained when biodegradable material LDH@SiO2 was chosen as drug carriers, ND DNA vaccine pVAX1-optiF was selected as the model drug through a coprecipitation method of drug encapsulation. The physicochemical characters, stability, biological activity, cell toxicity and immune effect of the pFDNA-LDH@SiO2-NPs were evaluated. The results demonstrated that:1) The LDH@SiO2 had been synthesized with morphous regulation, integrated profile, smooth surface, suitable size. The average particle size of the LDH@SiO2 was 477.2 nm with low polydispersity index 0.005 and proper Zata Potential+9.71 mV;2) Bacteriostatic test results showed that the LDH@SiO2 nanoparticles have different inhibitory effect on the Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Candida albicans;3) We determined the optimization conditions of pFDNA-LDH@SiO2-NPs:6.3 g NaOH,9.1 g NaNO3 and 0.4 g pFDNA-SiO2-NPs were dissolved together in deionized water (total volume,72 mL).0.06 M Mg(NO3)2·6H2O and 0.03 M Al(NO3)3·9H2O were dissolved together in deionized water (total volume,63 mL), and then were dropwise added in the above solution slowly and stirred at the speed of 3 mL/min at room temperature for 2 h. All the operations were done under N2 atmosphere to avoid oxidation. The expected pFDNA-LDH@SiO2-NPs were rinsed deionized water and dried at room temperature.4) The results demonstrated that the pFDNA-LDH@SiO2-NPs had been produced with morphous regulation, integrated profile, smooth surface, suitable size. The average particle size of the pFDNA-LDH@SiO2-NPs was 470.1 nm with low polydispersity index 0.005 and proper Zata Potential+16.82 mV. The entrapment efficiency was (92.58±0.89)%(n=5), drug-loading rate was (46.51±1.54)%(n=5);5) The release behavior of pFDNA-LDH@SiO2-NPs test in vitro demonstrated that pFDNA-LDH@SiO2-NPs release was a continuous release process after initial burst release;6) The stability test showed that the stability of pFDNA-LDH@SiO2-NPs was good in different pH NaOHsolutions, ionic concentration NaCl solutions and temperature;7) The indirect immunofluorescence results showed that the preparation method of pFDNA-LDH@SiO2-NPs didn’t destroy the biological activity of plasmid DNA, which had expressed in vitro;8) The pFDNA-LDH@SiO2-NPs mucosal delivery system was low toxicity and excellent safety, which could be proved by a cytotoxicity test;9) The SPF chicken vaccination results showed that mucosal immunity by pFDNA-LDH@SiO2-NPs used, the IgG and IgA contents of mucosal immune response positions were higher than intramuscular injection. In addition, the IFN-y, IL-2, IL-4 concentrations of serum and Lymphocyte transformation rate after immunization had been rised. the IgG, IFN-y, IL-2, IL-4 concentrations of serum of mucosal immunity kept rising, and and protective efficacy was superior to intramuscular injection.In a word, the result proved that nanoparticles mucosal delivery system not only induced immune responses in mucosal location but also produced high systemic humoral and cellular immune responses.ND DNA vaccine encapsulated in LDH@SiO2 nanoparticles had been assembled successfully with a further evaluation of its immunity effectiveness, and the mucosal delivery system had implemented a mechanism of the long-term mechanism of action in vivo, which could protect antigen form degradation, increase the vaccination methods, simplify the process of vaccination, decrease the vaccination times, enhance the immune effect. At the same time, this study also developed a safe, efficient and better performance gene delivery vector. It possessed a certain academic research value, potential popularization and application prospects for Newcastle disease prevention and control.