Construction Of ADV Whole Gene Optimized Nucleic Acid Vaccine And Immune Effect Preliminary Study

Aleutian disease of mink(Aleutian disease AD)is composed of Aleutian mink disease virus(Aleutian disease virus,ADV)a violation of mink immune system disease,also known as plasmacytosis.Because of its special pathogenesis,cause there is no effective prevention and treatment of mink Aleutian disease vaccine.In order to develop the effective vaccines of Aleutian mink disease,in this experiment,the DNA sequence of the isolated virulent strain of Aleutian mink disease virus was used as a template,the 428-446 amino acid sequence of VP2 gene was removed by overlap extension PCR technique and connected with pcDNA3.1(+)carrier,constructed the of full gene mutation recombinant plasmid pcDNA3.1-ADV-428 At the same time,based on the removal of 428-446 amino acid sequence of truncated 487-501 amino acid sequence,to construct the full gene mutation recombinant plasmid pcDNA3.1-ADV-428-487.The recombinant plasmid was transfected into L929 cells and the expression was detected by indirect immunofluorescence(IFA)assay.To select healthy mice immunize with the recombinant plasmid by intramuscular injection,detect antibody by indirect ELISA method and detect the CD3+,CD4+ and CD8+T lymphocyte subsets by flow cytometry in spleen cells from immunized mice at forty-second days;in serum of immunized mice IL-4,IL-10,IL-12 and IFN-? the content of 4 kinds of cytokines were used detected by ELISA kit.The results of the experiment showed that the two recombinant plasmids had good immunogenicity and immunogenicity.The above two kinds of nucleic acid vaccine constructed by intramuscular injection vaccination to mink body immune.Mink were attacked poison when after the completion of immunization,then every two weeks for blood collection of mink fingertips.the whole blood CD8+ lymphocyte subsets detection by flow cytometry;Detection of antibody levels in serum by indirect ELISA;Serum protein electrophoresis was used to detect the ? globulin percentage of total protein in serum and the application of polyethylene glycol precipitation determination of antigen antibody complexes in serum of mink.Through the above experimental determination pcDNA3.1-ADV-428 and pcDNA3.1-ADV-428-487 have effect on the prevention and cure of Aleutian disease of mink.Finally,the experimental results show that the data of pcDNA3.1-ADV-428 and pcDNA3.1-ADV-428-487 were better than those of the control group,the protective effect of pcDNA3.1-ADV-428-487 is better than pcDNA3.1-ADV-428,two nucleic acid vaccines have shown good vaccine potential.