| Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), causes immunosuppression in young chickens by destroying the precursors of B lymphocytes in the bursa of fabricius. In recent years, antigenic variant IBDV strains and very virulent IBDV (vvIBDV) strains have been emerging and caused considerable economic losses to the poultry industry around the world. The traditional inactivated or attenuated vaccines could not provide enough protection against these variants and vvIBDV. DNA vaccines which can induce humoral immunoresponse and cellullar immunologic response have been a focus of vaccine researches. 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.Our previous experiments have indicated that the polyprotein of IBDV had good immunogenicity and could induce satisfied protection. Chicken interleukin 18 (ChIL-18) can be used to enhance the immunogenicity of IBDV DNA vaccine as a molecular adjuvant and cooperate with IBDV VP2/4/3 in biological activity. In order to improve the immunity effect of polyprotein (VP2/4/3) DNA vaccine, fusion genes fragments(VP2/4/3-ChIL-18, ChIL-18-VP2/4/3) containing IBDV polyprotein (VP2/4/3) gene and ChIL-18 gene were constructed using the technique of splicing by overlapping extension (SOEing). Fusion gene fragments were directly cloned into pCI, an eukaryotic expression plasmid, to obtain the recombination expression plasmids pCI-VP2/4/3-ChIL-18 and pCI-ChIL-18-VP2/4/3. Then, co-expressing plasmid co-pCI-VP2/4/3-ChIL-18 which had two independent transcription units and could expressed VP2/4/3 and ChIL-18 respectively in one plasmid was constructed on the basis of pCI-VP2/4/3 and pCI-ChIL-18 stored in our lab. These recombination plasmids were transfected into Vero cells respectively introduced by lipofectamine 2000 reagent. The interest proteins were detected by indirect immunofluorescence, indicating these fusion genes were correctly expressed in Vero cells and the expressed proteins had immunoreactivity.The two-week-old SPF chickens were randomly divided into 6 groups and every chicken were immunized with recombination plasmids 200μg:group A received of pCI-VP2/4/3-ChIL-18, group B received pCI-ChIL-18-VP2/4/3, group C received co-pCI-VP2/4/3-ChIL-18, group D received pCI-VP2/4/3, group E received each of pCI-VP2/4/3 and pCI-ChIL-18, group F served as the unvaccinated control. All of the chickens were immunized intramuscularly at 2 weeks of age, and the chickens were boosted with plasmids using the same route and dosage after two weeks. The immunogenicity was compared among each testing groups though observing lymphocyte proliferation response of peripheral blood, detecting anti-IBDV neutralization antiby and ELISA antibody level and the protective immune responses of each group. The results indicted that the co-pCI-VP2/4/3-ChIL-18 could produce the best immune protection responses in these recombination plasmids, the pCI-VP2/4/3-ChIL-18 was in the second place and there were not significantly difference between pCI-ChIL-18-VP2/4/3 and pCI-ChIL-18+pCI-VP2/4/3, which was better than the pCI-VP2/4/3 was used alone.In conclusion, fusion genes plasmids of pCI-VP2/4/3-ChIL-18、pCI-ChIL-18-VP2/4/3 and co-expressed gene plasmid of co-pCI-VP2/4/3-ChIL-18 were constructed successfully in this study and expressed correctly in Vero cells and the expressed proteins had immunoreactivity. ChIL-18 and VP2/4/3 could cooperate to enhance the immune effect of IBDV polyprotein DNA vaccine. Comparing with vaccinating IBDV DNA vaccine and ChIL-18, using the recombination plasmids to inoculate animals have more advantages, such as injection only once, operation more convenience and low cost. It also provided basis data for the research of immune-enhancement of ChIL-18 and manufacture high effective and inexpensive DNA vaccination against IBDV. |
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