Immune Protection Effect Experiment Of Newcastle Disease Virus,Avian Influenza Virus And Infectious Bursal Disease Virus Recombinant Proteins

Newcastle disease,avian influenza and chicken infectious bursal disease are highly infectious diseases,affecting a variety of poultry and wild birds to varying degrees.It has caused great losses to commercial chicken and scattered chicken farmers.Reducing the number of chickens infected with viruses is an important first step in improving their productivity,and the use of vaccines is the most important way to control these diseases.At present,the products on sale in the market are only new stream inactivated vaccine,there are low immunization efficiency,need repeated inoculation and other problems.This laboratory prepared novel novel recombinant protein(HN,NP,M,F);avian influenza recombinant protein(HA,NA,M1).At the same time,the expression carrier p RSFduet1-sumo-VP2(BC6/85)of the IBDV VP2 protein was constructed and expressed in the E.coli system.In order to verify the immunoprotective effect of the recombinant protein,the first preexperiment was conducted through a single recombinant protein to verify the immunogenicity of the recombinant protein.Triple antigen solution was prepared according to the test results,and the serum antibody level of the chicken colony was detected.In the fourth week of immunization,two groups of NDV SZ strain and infectious French sac BC6/85 were used.The main research results of this paper are as follows.1.Expression and Test of Infectious cystic VP2 ProteinThe amplified VP2 gene was cloned to the p RSFduet1-sumo carrier to obtain the p RSFduet1-sumo-VP2 recombinant plasmid.After sequencing identification,the recombinant plasmid was converted to Escherichia coli BL21(DE3),and the expression of VP2 protein was induced by IPTG.The VP2 protein was purified using affinity chromatographic nickel column and the sumo label was cut.The recombinant protein products were analyzed by SDS-PAGE to obtain the target protein strip.Western-blotting further identified VP2 protein as responsive to IBDV positive serum.The AGP effect of VP2 protein was 1:64 as measured by the bidirectional agar diffusion experiment.The NDV recombinant protein produces titers of 2⁶ and the AIV recombinant protein of 2⁹.2.Immunogenicity test of recombinant proteinFirst,the expressed VP2 protein and the recombinant protein of novel disease and avian influenza were prepared with whitening adjuvant and SPF chickens at 30 days of age respectively.Then three proteins were emulsified to prepare antigen solution and immunize the chicken population.Blood collection was isolated on days 14,21,21 and 28 days after immunization,and the antibody valence was determined by blood coagulation suppression experiment and bidirectional agar diffusion experiment.The results showed that the chicken group HI titers was 3.4log2(NDV);7.9log2(AIV);VP2 in the immune group whose antibody peaked in week 4,with the highest AGP effect of 1:128.After 4 weeks of immunization,100%of chicken chickens received effective immunity.In triple antigen solution immunity experiments,80%of the chickens produced antibodies to Xincheng disease;90%produced antibodies to avian influenza;and 100%produced antibodies to infectious forensic cysts.3.Protective efficiency experiment of Newcastle disease virus and infectious bursal disease virus.28 days after triple recombinant protein immune chicken group SZ was injected 20?L(including 10^5.0EID50)and 0.2 m L(viral content greater than 100-BID).The results showed that NDV recombinant protein protection rate was 90%and IBDV VP2 protection rate was 70%.Summary:This study successfully expressed infectious sac VP2 protein,and prepared NDV(genotype?),Avian influenza(H9)and IBC6/85 triple antigen solution.Animal experiments showed that the recombinant protein IBDV VP2 had strong immunogen protection effect,It provides material and data support for the research and development of subunit vaccines of the subsequent new flow method genetic engineering.