Immune Potentiation By Genetic Immunization Of Interleukin Ⅱ From Xiaoshao Chicken

Interleukin 2(IL-2) is a lymphokine produced by activated T lymphocytes which exerts immunoregulatory effects on a variety of immune cells, including T cells, B cells, natural killer cells and lymphokine -activated killer cells. Utilization of IL-2 as an adjuvant for avain deseases is becoming feasible as chicken homologues of mammalian cytokine IL-2 genes become available. Many studies indicate co-admimistration of antigen with IL-2 could enhance hormonal and cellar immunity in chickens. The cDNA of IL-2 gene of Xiaoshai chicken was amplified and inserted into the prokaryotic vector pET-28b. The recipient E. coli cells BL21 were transformed with the recombinant expression vector pET-28b-IL-2. IL-2 was expressed under the induction of IPTG as identified by SDS-PAGE. An emulsion was prepared with the purified IL-2 and injected into rabbits. The polyclonal antibody in the rabbit serum is identified by ELISA and Western blotting.The cDNA of IL-2 gene of Xiaoshai chicken was amplified and inserted into the eukaryotic vector pCI. The resulting recombinant plasmid pCI-IL-2 was transformed by electroporation into attenuated Salmonella typhimurium ZJ111, which was then used to transfect the Vero cells. DNA blotting revealed that the IL-2 gene was integrated into the genome of Vero cells. There was expression of the IL-2 protein as shown by indirect immunofluorescent assay. The 14. 5KD and 18. 5KD bands of the IL-2 protein from lysate of Vero cells was identified by SDF-PAGE and Western blotting, indicating the reactivity of expressed proteins.Safty tests indicated that ZJlll/pCI-IL-2 was of low pathogenicity to mice. There were only few mice dead at a high dose of 109cfu , No mice died after immunization with doses of 107cfu and 108cfu. It did not cause any death or side effect on chicken orally at doses of 107cfu,108cfu and 109cfu. At five week after immunization.no Salmonella typhimurium cellswere recovered from the spleen and liver at or blow 108cfu. Only a few Salmonella typhimurium cells were recovered from the spleen and liver at 10 9cfu . The Salmonella typhimurium cells in feces decrease over time. ZJ111/pCI-IL-2 showed high stability in vitro and in vivo as determined by enzymatic digestion and PCR.Effects of IL-2 on immunogencity of the DNA vaccine pCI-VP2/VP4/VP3 was examined. Fourteen-day-old non-immunized chickens were co- adminis trated orally and intradermally with DNA vaccine and ZJlll/pCI-IL-2 at three dosage levels (107cfu , 108cfu , 109cfu),and boosted two weeks later. Control groups(challenge control, normal control, DNA vaccine control) were included. The chickens were challenged with virulent infectious bursal disease virus(IBDV) two weeks post-boosting. Co-administrated was more effective than the DNA vaccine alone in induction of antibody response and protection. Co-administration with ZJlll/pCI-IL-2 at 109cfu and 108cfu offered a protection rate of 81% against the challenge, while that at 10'cfu provided 75% protection. The protection rate was 62.5% for the DNA vaccine alone. Furthermore, proliferation responses of T lymphocytes from thymus and spleen, and of the B lymphocytes from bursa could be significantly enhanced by co-administration of DNA vaccine with ZJlll/pCI-IL-2. These results clearly indicated that chicken IL-2 was a strong adjuvant to potentiate immunogenicity of the IBDV DNA vaccine, A dose of lO'cfu of ZJlll/pCI-IL-2 seems to be enough for potentiation. Oral adjuvant is more effective than the naked plasmid pCI-IL-2 in potentiation of the DNA vaccine.In conclusion, The gene of IL-2 was expressed in prokaryotic and eukaryotic cells. Oral administration of ZJ111/pCI-IL-2 as adjuvant increased immunogenicity of the IBDV DNA vaccine. Oral delivery of the adjuvant to chickens is time-saving, cheap, convenient, and suitable formass immunization particularly in the poultry industry.