| As the specific receptor chain of interleukin 2(IL-2), Interleukin-2 receptorαlpha chain (IL-2Rα) was the specific surface marker of activated T cells, had an important role in the process of biological functions to result in by IL-2; interleukin 18(IL-18) was a new therapeutic agents and immune adjuvant which had broad prospect, also, could stimulate the expression of immunore- gulatory factor as IFN-γand IL-2,that enhanced the protection of the body.The goose Interleukin-2 receptorαlpha(GoIL-2Rα) cDNA was firstly cloned by 3′RACE and 5′RACE, special primers were designed according to chicken Interleukin-2 receptor alpha(ChIL- 2Rα) and duck Interleukin-2 receptorαlpha(DuIL-2Rα). This new cloned cDNA is composed of 1027bp, the precusor protein consisits of 240 amino acids, with 20aa signal peptide. The predicted protein shares of 87.08%, and 53.94% in amino acid sequence, compared with duck and chicken respectively. There are ten half-cystines in GoIL-2Rα, and eight were conserved in all the counterparts of poultry. Comparative analysis of the predicted GoIL-2Rαprotein with other IL-2Rαhomologous revealed that GoIL-2Rαhad a closer relationship with DuIL-2Rαthan that of mammals. Second structure analysis showed that there is a transmembrane domain(217-240) at the C-terminal. The goose Interleukin-18(GoIL-18) cDNA was firstly cloned by the same method, This new cloned cDNA is composed of 863bp, the precusor protein consisits of 200 amino acids, without signal peptide, also lacks the traditional N-Glycosylation sites,similar to mammals. There is a leader sequence in mammals, that can be cut at specific site by IL-1β-converting enzyme (ICE or Caspase-1). Comparative analysis of the predicted GoIL-18 protein with avian and mammalian reveales that there is 30aa leader sequence in precursor protein.The PCR product encoding the extracellular domain of the mature GoIL-2Rαwas subcloned into the prokaryotic expression vector pET-30a, than constructed recombinant expression vector pET-30a-mIL-2RαQ. The bacterial competent E.coli Rosetta(DE3)cells transformed with the recombinant vector was induced by IPTG. SDS-PAGE and Western blot analysis showed that a new band with an approximate molecular weight of 34kDa was readily observed, and was 19.4% of total bacterial protein, also it could be recognized by anti-His mAb. The recombinant protein expressed in the pET-30a system existed in inclusion form, and could be purified by Ni2+ affinity column. Using the GoIL-18 cDNA obtained by RT-PCR as template, the PCR product encoding the mature GoIL-18 protein was subcloned into the prokaryotic expression vectors pET-30a. Than constructed recombinant expression vector pET-30a-mIL-18.The bacterial competent E.coli Rosetta(DE3)cells transformed with the recombinant vector was induced by IPTG. SDS-PAGE and Western blot analysis showed that a new band with an approximate molecular weight of 28kDa was readily observed, and was 23.3%-33.3% of total bacterial protein which increased with time, so it could be recognized by anti-His mAb, he recombinant protein expressed in the pET-30a system existed in inclusion form. In order to conveniently detect the activity, than optimiized conditions of the expression in E.coli, mainly changed the induced temperature, amount of IPTG and medium composition, results showed that the supernatant had a small amount of target protein when the induced temperature was 25℃, the medium had 1%glycine and 1%TritonX-100.The product encoding the mature GoIL-18 protein was inserted into the multiple cloning sites of transfer vector pFastBacHTB to construct recombinant transfer vector pFastBacHTB-mIL-18, and transformed into competent bacteria DH10BacTM in order to recombinant in vitro, than transfected the recombinant bacmids into insect. Transfected Sf9 cells Morphology were observed and recombinant viruses were identified, indicated that recombinant rod particles had been transfer- red to Sf9 cells, and amplified in the cells. Positive serum was prepared by the rGoIL-18 protein expressed with prokaryotic system. Indirect immunofluorescence and Western blot analysis showed that rGoIL-18 had been expressed in Sf9 cells. Detecting IFN-γof stimulated lymphocytes supernatant antiviral activity by micro cytopathic inhibition assay in vitro, indirectly detecting the activity of rGoIL-18 expressed by two systems; using MTS kit to detect goose spleen lymphocyte proliferation, the results showed that: 1)The activity of IFN-γcould not be detected in the collected cells culture supernatant after induced 24h, but could be detected after induced 48h and 72h, there was not inhibitory activity of rGoIL-18 for Newcastle disease virus: 2)The rGoIL-18 protein expressed by two systems could stimulate lymphocyte proliferation(P<0.01), compared with other times, the stimulation index was highest at 48h, the difference was significant.The GoIL-2 and GoIL-2Rαwere connected into the vectors pCMV-Myc and pCMV-HA, than constructed recombinant vector pCMV-Myc-IL-2, pCMV-HA-IL-2Rα. The twoco-transfected CHO cells, than identified by RT-PCR and indirect immunofluorescence, indicated both were expressed in CHO cells. Immunoprecipitation studies showed that expected target band was not appeared, indicated that the affinity was low between GoIL-2 and receptorα, than using immunoprecipitation could not study the interactions. Preparing monoclonal antibodies of GoIL-2, identified the higher activity of monoclonal antibodies with the rGoIL-2 protein expressed by the insect cells, than preparing the ascites and purifying, at the same time, purifying the positive sera of rabbit anti GoIL-2. Using the purified ascites as capture antibody and the purified positive serum as detection antibody to establish GoIL-2 antigen capture ELISA, than detecting the GoIL-2 levels of lymphocyte supernatant stimulated by rGoIL-18. The GoIL-2 could be detected in the collected culture supernatant after induced 48h(P<0.01), also could be detected after induced 72h, the difference was significant compared with 24h. This showed rGoIL-18 protein could stimulate lymphocytes to secrete IL-2.
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