Exploration Of Ligand And Receptor Alpha For Duck Interleukin-2

Using a pair of special primers designed according to chicken interleukin 2 gene (ChIL-2) sequence and genome DNA template obtained from duck peripheral blood monocytes by simplified DNA isolation procedure, duck 1L-2 (duIL-2) gene was amplified by long distance-polymerase chain reaction (LD-PCR), with a full-length of 3528 bp by sequencing. The organization, promoter and the predicted protein structure of duIL-2 gene were analyzed by bioinformatics. Then the three dimensional structure of duIL-2 protein was further constructed. The results showed that duIL-2 gene had significant similarity with that of chicken and mammalian homologous, with typical four exons and three introns. There were remarkable conserved domains in duck , chicken and mammalian IL-2 promoters, including the transcription factor binding sites AP-1, NF-AT, CD28RE and OCT, the TATA box and the predicted transcription start site. The predicted duIL-2 ORF was 420 bp, encoding a 140 aa prepeptide. Comparative analysis of the predicted duIL-2 mature protein with other IL-2 homologous revealed that duck IL-2 had a closer relationship with goose and chicken IL-2s than that of mammals. The predicted duIL-2 is a typical four alpha-binds structure.Using the duIL-2 cDNA obtained by RT-PCR as template, the PCR product encoding the mature duIL-2 protein was subcloned into the prokaryotic expression vectors pBAD/HisB and pET28a. The bacterial competent E. coli LMG194 and BL21 (DE3) cells were transformed with the pBAD/HisB-duIL-2 and pET28a-duIL-2 plasmids, respectively, and then were induced with L-(+)-arabinose and IPTG respectively. Western blot showed that new bands with approximate molecular weight of 18.7 KDa and 14.6 KDa were readily observed on Coomassie blue-staining gels. The aimed protein bands could be recognized by anti-Express and anti-His monoclonoal antibodies, respectively. Using the rduIL-2 protein expressed by pET28a-duIL-2 system, two strategies were tried to acquire refolded rduIL-2: by dilution-dislysis method from the purified rduIL-2 monomer under denatured conditions; by easy oxidization from the FPLC purified rduIL-2 monomer under nature condition. The refolded products from the former existed in monomer and multimer forms, with little yield. However, high yield of refolded rduIL-2 monomer could be obtained from the the latter procedure. PF-2D analysis showed that the refolded rduIL-2 monomer has a PI of 4.3, with no potential isomers. The refolded rduIL-2 from the first strategy resulted in visible flocculent precipitate when stored at 4℃within a week. However, the refolded monomer from the second strategy was stable under the same condition.Five hybridoma cell lines secreting monoclonal antibodies (mAb) against duck IL-2 (named as 1H4. 2B3. 4G12. 5F6 and 5H6) were produced by fusing mouse myeloma ceils (SP2/0) with spleen cells from BALB/c immunized with the purified recombinant duck IL-2 protein. Among the five mAbs. 5H6 belongs to IgG2a, and the other mAbs were of the same isotype of murine antibodies igG1. The five murine antibodies' light chain hold the same isotype k. Western blot analysis showed that the five mAbs could react with duck IL-2, mAb 5H6 could also react with chicken IL-2. Immunochemistry analysis showed that mAbs 2B3, 4G12, 1A4 and pAb against prokaryotic expressed duIL-2 could recognize the duIL-2 expressed in Vero cells. Inhibition assay indicated that the mAb 2B3 significantly inhibited the proliferation of Con A -stimulated duck SMC enhanced by duIL-2.Using the refolded duck interleukin-2 monomer as bioactive standard, series of in vitro and in vivo bioactivities of rduIL-2 were investigated: The in vitro lymphocytes proliferation ability of rduIL-2 was detected by MTT method; then the in vivo function of rduIL-2 was investigated by iv administration of rduIL-2, using chicken as model; the potential side effects of rduIL-2 were firstly investigated by high doses of sequential administration, using duck as animal model; finally, we explored the potential adjuvant effect of duck IL-2, by intravenous injection of rduIL-2 protein. Results showed that the prepared duIL-2 could enhance the proliferation of Con A stimulated duck SMC, with a dose dependent manner. High dose of rduIL-2 resulted in side effects such as fever and inflammation. Low dose of rduIL-2 up-regulated in vivo the amounts of CD4~+ T cells, instead of CD8~+ T cells. The frequency of CD4~+ T cells showed no correction with the rduIL-2 dose in the dose range (0.1-25μg). Low dose of rduIL-2 showed significant adjuvant effect in AIV H5N1 vaccination. However, high dose rduIL-2 inhibited the effect of AIV vaccine.The duck interleukin-2 receptor alpha (duIL-2Ra) cDNA was firstly cloned by RACE combined with homologous cloning strategy. This new cloned cDNA is composed of 886 bp. Massive data were acquired by bioinformatics analysis: The precursor protein consists of 226 amino acids, with 20 aa signal peptide; The predicted mature protein shares identity of 15-25%, and 61% in amino acid sequence, compared with the counterparts of mammals and chicken respectively; There are 23 aa sites conserved in all the compared species. There are eight half-cystines in duIL-2Rα, and 7 were conserved in all the compared IL-2Ra. An interesting thing is that during the evolution, aa insertion instead of depletion occurred: 1, 3, 1, 26-29, and 9-16 aa inserted in the sites between P24-P25, N90-A91, P111-K112, I185-R186, and in the C terminus. This insertion phenomenon might be the results of evolutional pressure of complex immunologic functions of IL-2Rα. Second structure analysis showed that there is a transmembrane domain (182-206) at the C-terminal.The PCR product encoding the extracellular domain of the mature duIL-2Ra was subcloned into the prokaryotic expression vector pET32a. The bacterial competent E. coli BL21 (DE3) cells transformed with the pET32a was induced by IPTG. SDS-PAGE and Western blot analysis showed that a new band with an approximate molecular weight of 40 KDa was readily observed, and could be recognized by anti-His mAb. The recombinant protein expressed in the pET32a system existed in inclusion form. The yield of duIL-2Ra expressed in E. coli BL21 (DE3) cells was about 32mg mg / 3g bacterial, when analyzing the preparation of duIL-2Rαinclusions.Fifteen hybridoma cell lines secreting monoclonal antibodies against rduIL-2Rαwere produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the purified rduIL-2α, namely 1A4,3B6, 7C8, 1C5, 3D1,1E4, 2E1, 6E1, 6E9, 2F4, 6F2, 4G10, 3H11, 4H3 and 6H7. Ig class and subclass of all the fifteen mAbs were IgG1,κ. Western-blot analysis showed that the fifteen mAbs could react with rduIL-2α. ICC showed that five mAbs could recognize duck fusion IL-2Rαtransiently expressed in Vero cells. Further using Con A activated duck SMC as endogenous CD25 antigen, mAbs 3H11and 6F2 were shown to recognize the endogenous duIL-2α.Using avian flu virus subtype H9N2 infection as model, the mAb 6F2 against duIL-2Rαas capture antibody, we analyzed the kinesis of DuCD25~+ cells in virus infected ducks, by FACS method. Results showed that the frequency of DuCD25~+ cells in normal ducks is very low(<4%). However, in the in infected ducks, the frequency of DuCD25~+ cells reached the peak within 48h post infection, then declined to normal 144h post virus infection. These results indicated that the DuCD25~+ cells played an important role in the immune response against virus infection.