Research On Immune Adjuvant Of The Recombinant Plasmid Encoding Chicken Interleukin 2 (pCI-ChIL-2)

Interleukin-2 (IL-2) is a lymphokine produced by T lymphocytes following antigen stimulation. This lymphocyte is recognized as an important immuno-regulatory molecule and plays roles in T lymphocyte proliferation, B lymphocyte development, and natural killer (NK) cell activation. Chicken interleukin-2 (ChIL-2) was firstly acquired by Sundick in 1997, subsequently some experiment proved that it had similar immune function with mammalian IL-2 in vivo and in vitro. As an important immuno-regulatory molecule, IL-2 could enhance hormonal and cellar immunity in chickens when co-admimistration of antigen. With the increasing application of ChIL-2, more information is desireable to know. In present study, the security and application of ChIL-2 are elucidated detailedly.The ChIL-2 gene cloned and saved in our lab was cloned and conserved in pGEM-T-Easy vector. The full open reading frame (ORF) of ChIL-2 was amplified by PCR. Restriction sites for EcoRI and SalI were introduced into the primers and the stop codon of ChIL-2 gene was removed from the downstream primer. The PCR product was inserted into eukaryotic expression vector pCI, resulting in pCI-ChIL-2. The EGFP (enhanced green fluorescence protein) gene, released from the pEGFP-N1 vector by SalI and NotI digestion, was further introduced into pCI-ChIL-2, resulting in pCI-ChIL-2-EGFP, which encodes a fusion protein ChIL-2-EGFP.To investigate the fusion protein expressed in vitro, Vero cells, CEF and PBLC were transfected with pCI-ChIL-2-EGFP and pCI respectively. The expression of ChIL-2-EGFP fusion protein was found in cytoplasm and nucleus; the fluorescence appeared at 8 h p.t. (post transfection) and peaked at 36 h p.t. Over 70% of Vero, 50% of CEF and 80% of PBLC were positive in all samples at 36 h p.t. The cells transfected with pCI did not exhibit any significant fluorescence. pCI-ChIL-2-EGFP was investigated for its expression after intramuscular (i.m.) injection in chickens. The expression profile of the fusion gene (ChIL-2-EGFP) in vivo was measured by RT-PCR, ELISA and fluorescence microscopy. The EGFP expression was detected from 8 h p.i. to 14 days p.i. and peaked at 5 days p.i., when the number of EGFP-expression myocytes was about 5% in the injected site. These results demonstrate that intramuscular administration of plasmid DNA leads to long-term expression in vivo and in vitro.Distribution of pCI-ChIL-2 in chickens was investigated after intramuscular (i.m.) injection. After the i.m. injection, serum distribution was detectable from 2 h post inoculation (p.i.), peaked at 8 h p.i., and disappeared at 7 days p.i. The plasmid DNA could be observed in organs including heart, liver, lung, spleen, bursa and inoculated muscle at different time points. 32 days p.i. the plasmid DNA was undetectable in any organ except inoculated muscle.In order to investigate the immnoadjuvant effect of ChIL-2, pCI-ChIL-2 was co-administrated with H5 subtype inactivated avian influenza virus (AIV) vaccines. Non-immunized commercial chickens were inoculated initially at 25-day-old and boosted 2 weeks later. Proliferation responses of chicken periphery blood lymphocytes were measured by MTT assay and the titers of specific antibodies to the H5 vaccine by HI assay. The cellular and humoral immune responses were significantly enhanced in the groups co-immunized with pCI-ChIL-2 or rChIL-2 compared to the group inoculated with H5 vaccine only (P < 0. 05). Peak values of MTT and HI in the rChIL-2 co-inoculated group were attained 4 weeks after initial inoculation, which was earlier than the group co-inoculated with pCI-ChIL-2. These results presented evidence that both pCI-ChIL-2 and rChIL-2 could be employed as effective immunoadjuvant to conventional vaccines against avian influenza.With the warrant from the ministry of agriculture, the midst trial, the environment release trial, and production trial of pCI-ChIL-2 plasmid were carried out successively. Results showed that intramuscular administration of plasmid DNA leads to widespread and long-term distribution. After administration, the ability of microbe in environment against Amp was not changed and the chickens were in good circumstance. Detection of antibody showed that pCI-ChIL-2 could enhance the immune effectivity of commercial vaccines significantly.To sum up, the distribution of plasmid encoding ChIL-2 is widespread and the expression is long-term in chickens after intramuscularly administration. pCI-ChIL-2 plasmid is an effective adjuvant to avian vaccines. These findings provide theoretical base for applying eukaryotic expression plasmid encoding ChIL-2 as an adjuvant in the poultry industry.