The Study On Nucleic Acid In Situ Gel Vaccine Of Newcastle Disease

Newcastle disease is one of the most serious disease among the poultry industry caused by Newcastle disease virus(NDV).The F protein as a protective antigen is required for NDV infected cells.F genes are cloned and connected to expression vector.Poloxamer thermosensitive in situ gel is flowing fluid at room temperature and can flood into the gaps between tissues after injection,but it forms semisolid gel at the site of injection as a result of temperature.Therefor the Poloxamer thermosensitive in situ gel can prolong the release of drug.In this study,recombinant plasmids were added into poloxamer solution to research for the effectiveness of immunity in chicks and to evaluate the sustained-release effect on nucleic acid vaccine through in situ gel method which provided theoretical basis for clinical application.1 Construction of recombinant plasmid of F gene of NDVThe standard virulent virus of Newcastle disease(F48E9)was inoculated into SPF chick embryo,then the RNA of virus was extracted and target fragment was amplified.The vector pVAXl with target fragment were transformed into E.coil DH5a competent cell.The result showed that the target fragment of a virus amplified by PCR was about 1600 bp.The target fragment connected to pVAXl has two bands at 1600 and 3000 bps by enzyme digestion and its nucleotide size is 1662 bp by the sequence analysis which is consistent with the expected results.The recombinant-plasmids can be expressed in competent cells.2 The preparation of nucleic acid in situ gel vaccine of NDVThe appropriate blank prescription of poloxamer thermosensitive gel were selected through the research on GT,Gt,viscosity and pH.Then the recombinant plasmids were added into the prescription to detect the nature of poloxamer.The final selected blank prescription was 16%:2.5%(P407:P188),the GT,Gt,viscosity and pH were 36.29 ℃、50.36 s、9.99 s、6.46,and the final selected blank prescription was stable after repeated freezing and thawing and treatment through high pressure sterilization.The recombinant plasmid with different concentration had little effect on the GT and pH,but the Gt and viscosity increased.However the nature of the reverse thermal gel solution did not affect.The result showed that the addition of recombinant plasmid in poloxamer is successful for forecast of NDV vaccine..3 Study on the immune validity of nucleic acid in situ gel vaccine of NDV140 14-day-old chickens were assigned to 7 groups(4 groups:the healthy control,blank control,immune control,plasmid control and 3 groups of different concentration of nucleic acid in situ gel vaccine)randomly.The chickens except blank control group and healthy control group were vaccinated with different vaccines and repeated vaccination at 28 days old for immune control group and plasmid control group.On days 7,14,21 and 28 after the first vaccination,the peripheral lymphocytes proliferation and serum ND antibody titer were determined and at 49 days the test groups were challenged to evaluate the protective effect of vaccine.The results showed that the nucleic acid gel vaccine could effectively promote peripheral blood lymphocyte proliferation and antibody titer at high,medium,and low doses.But after the attack of NDV in chickens,only the high and medium dosage groups had protective effect.The nucleic acid in situ gel vaccine of Newcastle disease can improve the immune function of chicken in a certain extent and have sustained-release effect.This sustained-release effect is important factor for developing the immunity during application of vaccines.