| Interleukin-18(IL-18), a novel discovered cytokine, was first described as an interferon-gamma-inducing factor(IGIF) primarily by its ability to induce IFN-γproduction in T cells and natural killer(NK) cells. However, with more and more research of IGIF, it was found that IGIF has its constitution and many biological functions. So in 1996 it was named as Interleukin-18. IL-18 is mainly secreted from cells of monocytemacrophage system. Previous studies were related to the detection of human and various others mammals IL-18 by gene cloning, expression and biological activity. It has been demonstrated that IL-18 exists in cells of human, mammal and some kinds of birds. Although IL-18 is structurally homologous to IL-1, its function is related to IL-12 and they have synergistic actions. IL-18 not only can enhance Th1-mediated responses, but also has the capacity to stimulate Th2-mediated responses. It shows that IL-18 plays an important role in host defence against various micro-organisms infection and tumor. Cytokines including IL-18, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics in control of diseases in intensive livestock and poultry industries. The discovery of chicken interleukin-18 gene is very meaningful for both the study on comparative immunology and the therapeutics for poultry diseases.In this study,one pair of specific primer for mature chicken interleukin-18(ChIL-18) cDNA was designed and synthesized according to the previously published gene sequence of ChIL-18.The full length cDNA gene of ChIL-18 encoding mature active protein was amplified from LPS–stimulated MDCC-MSB1 cells by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Then it was cloned into pMD18-T vector. Sequencing analysis showed that the nucleotide sequence of this ChIL-18 mature protein gene was 5l0bp including the stop coden and the same as the published ChIL-18 cDNA sequence by Schneider K.To highly express chicken interleukin-18 in E.coli and purify the fusion protein, cDNA fragment encoding the mature ChIL-18 was subcloned into the downstream of GST gene according to the right open reading frame(ORF) in the prokaryotic expression vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli BL21(DE3) strain, and then induced by IPTG at 37℃. The recombinant ChIL-18(rChIL-18) was expressed efficiently in forms of inclusion body with the yield accounting for 32% total bacteria protein. Optimizing select the parameters of expression, the level of expression of rChIL-18 is highest when IPTG reaches 0.2 mmol/L and induced time is 4 hours. SDS-PAGE and Western-blotting validated the expression of mChIL-18, and showed that the recombinant fusion protein had a molecular weight approximately 44 kDa. Expressed product tagged by GST was purified with affinity chromatography. The expressed specific band was excised from the gel and injected into mice once two weeks for 3 times, then we collected the antiserum from the mice.Examine the expressed protein by Western-blot with the antiserum against rChIL-18 showed the in vitro expressed protein of mChIL-18 has its immunology activity. At last, we constructed pcDNA3.1TOPO-mChIL18 eukaryotic expressing plasmid, which contains mChIL-18 gene. The plasmid was transfected into Chicken Embryonic Fibroblast(CEF) by lipofectamine reagent. 24,48,72 hours later after transfection, we testified the expression of mChIL-18 by Indirect Immunofluorescence Assay(IFA) with the antiserum of mChIL-18 expressed in E.coli. The tests justified the expression of mChIL-18 in prokaryotic and eukaryotic expressing systems.As a result, we laid a solid foundation and prepared experimental material for the future studies on the bioactivity of ChIL-18.
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