Preparation Of DNA Genotype Ⅶ DNA Nanoparticle Vaccine

Newcastle Disease（ND）is an acute,highly contagious disease caused by Newcastle Disease Virus（NDV）,which is mainly caused by infected chickens and turkeys.At present,the epidemic strain of Newcastle disease virus is a strain of type VII,especially the strain of VIId subtype strain has caused huge economic losses to the poultry industry in China.Compared with traditional vaccines,DNA vaccines have the advantage of being safe and inducing high levels of cellular and humoral immunity,but the immunological effects of intramuscular injection alone are not ideal due to the presence of nucleases and the like.The nano-mucosal immuno-delivery system utilizes nanotechnology to improve the effectiveness of existing vaccines with biodegradable,non-polluting nanomaterials.There are few studies on the combination of Newcastle disease DNA vaccine and nanomaterials.Therefore,the biomaterial chitosan（CH）,sodium tripolyphosphate（TPP）and Mg SO₄are used as carriers to Newcastle disease virus gene.The F gene eukaryotic expression vector p CAGGS-F of type VII strain（r GM）was used as a model drug,and DNA nanoparticles were prepared by ion cross-linking method,and the immune effect was analyzed.The main contents are as follows:The F gene of Newcastle disease virus type VII strain（r GM）was cloned into p CAGGS to construct eukaryotic expression plasmid p CAGGS-F,and mixed with biomaterial chitosan,TPP and Mg SO₄to prepare Newcastle disease virus vaccine.Type VII DNA nanoparticle vaccine,both of which can express the corresponding F protein on DF-1cells.Through the optimization of different parameters,in the single factor experiment,the DNA nanoparticles prepared by multi-ion cross-linking have greater stability in the range of p H 4.5～6.5,and the particle size is smaller and decreases first and then rises.The trend of Zeta potential is decreasing.At p H 6.0,the nanoparticles are round in TEM,the particle size is small,the dispersion is good,and there is no adhesion.The particle size is 214.8±7.4 nm,and the zeta potential is 20.2±1.6 m V,PDI is 0.204.Select other optimal parameters,stirring speed 1 000 r/min,stirring time 30 min,centrifugal speed 18 000 r/min,centrifugation time 30 min,ultrasonic crushing power 200 W,crushing time 15 min for orthogonal test optimization,screening other main influencing parameters,preliminary The optimal conditions were determined to be chitosan concentration of 1 mg/m L,DNA concentration of 500 ng/μL,Mg SO₄concentration of 1 mg/m L,and TPP concentration of 1mg/m L.Under the condition that the overall mass ratio of CH:Mg SO4:TPP:DNA is 5:1:2:1,the DNA nanoparticles prepared according to the optimized conditions have no obvious voids on the surface,and have a well-defined circular shape with good Dispersion,and no more aggregation or sedimentation damage,the average particle size of the particles in the nanoparticle solution is 154.8±7.4 nm,the zeta potential is 21.0±2.1 m V,and the encapsulation efficiency of the nanoparticles is 92.1±0.16%.Anti-DNase I test results show that the prepared DNA nanoparticles can effectively protect DNA from nuclease degradation;in vitro release test indicates that the release of DNA nanoparticles is a process of slow release after burst release,14 days of DNA The cumulative release was 87.9%.DF-1 cells were used for MTT assay.Compared with the control cells,no obvious changes in cell morphology were observed.The cell viability was（90.05±1.42）%.In the overdose immunization test,the immunized chickens did not abnormal or sick,this results showed that the chitosan-TPP mucosal immune delivery system and the prepared DNA nanoparticle vaccine have high biosafety.The storage stability test showed that the average particle size of the DNA nanoparticle vaccine freeze-dried group and the 4℃nanoparticle solution group and PDI did not change significantly within 28 days of storage,which was stable.In the immune effect test:in the minimum immunization dose test,different doses of Newcastle disease virus gene type VII DNA nanoparticle vaccine were immunized by eye drops,and 100μL of 10³EID₅₀Newcastle disease r GM virus was used for the test group two weeks after the second immunization.Chickens underwent eye-dropping nasal challenge,100μg group and 200μg group had 100%protection rate,no Newcastle disease symptoms,but considering the immune stimulation and cost,finally determined that 100μg is the minimum immune dose.The control group and other immune dose groups had different degrees of onset and death,and the protection rate was not high.Compared with the naked plasmid group,the nanoparticle vaccine group was more capable of stimulating mucosal immune response during the immunoassay and higher Ig G,Ig A antibodies;In the challenge protection test,the DNA nanoparticle vaccine group（i.n）has a 100%protection rate,has a good protective effect,the results show that the vaccine has a control against Newcastle disease Certain application value.This experiment initially explored the application of Newcastle disease vaccine,and provided some new ideas for the development of new Newcastle disease preparations.