| Infectious bursal disease virus(IBDV) can cause infectious disease with the character of immunosuppression and large scale of loss due to higher susceptibility to other pathogens and immunization defeat. Classic method of inactive vaccine and low-virulence vaccine have been confronted with the threat of variant strains and very virulent strains of IBDV isolated since 1980's. DNA vaccine, which can induce all-round immunity, has been a focus of vaccine research. However, immune effect of DNA vaccine is not ideal, so how to enhance the Immunogenicity of DNA vaccine has been the key to recent studies.Chicken interleukin 2(ChIL-2), as a molecular immune adjuvant, can be used to enhance the immunogenicity of IBDV DNA vaccine. At the same time, ChIL-2 can cooperate with IBDV VP2/4/3 in biological activity. In prophase work, we have make sure that pCI-VP2/4/3 has good protection efficacy and chicken interleukin 2 can enhance the immunogenicity of IBDV DNA vaccine clearly. Recombinant eukaryotic expression vectors, which contain fusion genes of infectious bursal disease virus (IBDV) polyprotein (VP2/4/3) gene and chicken interleukin 2 (ChIL-2) gene, were constructed by using the technique of splicing by overlapping extension (SOEing) and three times PCR. We have used two modes of gene fusion of IBDV VP2/4/3 and ChIL-2 because the factors of expression and secretion of fusion genes in eukaryotic cell is indistinct. So ChIL-2 was placed at C end and N end of VP2/4/3 respectively. Fusion gene fragments were directly cloned into pCI, an eukaryotic expression vector, to get recombinant expression plasmids pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3. In this way, ChIL-2 and IBDV VP2/4/3 genes can be expressed in the same cell, and ChIL-2 can exert adjuvant effection highly. Otherwise, we used the primers containing a linker which is composed of 15 low hydrophobe and low charge amino acids so that the expression products of fusion genes can be close to nature state.The recombinant plasmids were transfered into Vero cells introduced by Lipofectamine2000 reagent. Gene specific RT-PCR showed that the fusion genes were transcribted in Vero cells. Indirect immuno-fluorescent assay(IFA) showed that the expressed protein was immune-reactivity with chicken anti-IBDV serum and anti-ChIL-2 serum respectively. By SDS-PAGE and Western blotting, 60KD, 56KD and 30KD special bands ofexpression product of pCI-IL-2-VP2/4/3 can be identified respectively in cell fragmentation which have been thansferd after 72 hours. At the same time, 48KDn 44KD and 42KD special bands of expression product of pCI-VP2/4/3-IL-2 can be identified respectively in the same condition, but VP4 protein haven't been detected at all. Incubating with rabbit anti-IBDV serum as primer antibody, western blotting showed that 60KD, 56KD and 30KD special bands of expression product of pCI-IL-2-VP2/4/3, corresponding to the molecular weight of IL-2/VP2 precursor protein, mature protein and VP3 .This result suggests that the fusion genes are expressed correctly in Vero cells. Whereas incubating with rabbit anti-ChIL-2 serum as primer antibody, western blotting showed that 44KD and 48KD special bands of expression product of pCI-VP2/4/3-IL-2, corresponding to the molecular weight of VP3/IL-2 precursor protein and mature protein. This indicates that not only the fusion genes are expressed correctly in Vero cells but also the expression products have immune-activity. And the linker retains the nature state of fusion genes.Recombinant plasmids of pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3 are prepared for DNA vaccine. Fourteen-day-old non-immunized chickens were vaccinated intramuscularly with DNA vaccines, and boosted two weeks later. Anti-IBDVserum ELISA antibody and neutralization antibody were detected in designated dates. All test groups were challenged with reference virulent IBDV strain BC6/85 at 7th week and all chickens were killed after 3 days. Protection rate and bursal/body were computed, and histopathology detection was performed. These results indicated: 1)Recombinant plasmids of pCI-...
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