Stable Expression Of Chicken Interleukin-18in A Eukaryotic System And Generation Of Monoclonal Antibodies Against Chicken Interleukin-18

Interleukin18(IL-18) was identified as an interferon-gamma (IFN-γ) inducing factor (IGIF)previously, which acts in synergy with IL-12. IL-18plays an important role in innate and acquiredimmunity. IL-18can induce Th1, natural killer (NK) and NKT cells to produce IFN-γ and theproduction of IL-2, IL-8, GM-CSF and TNF-α. Chicken interleukin-18(ChIL-18) protein contains198amino acids and it has30%similarity with IL-18of humans and others mammals.Partial ChIL-18gene fragment without signal peptide was amplified by RT-PCR from the extractedRNA of conconavalin A (ConA)-stimulated peripheral blood lymphocytes (PBL), cloned into pET-30a(+) vector and then expressed in Escherichia coli (E. coli) BL21(DE3) cells. The expressed protein wasabout28kDa which mainly in the form of inclusion body and crude purified by cutting proteincontaining gel slices from SDS-PAGE and immunized in rabbits to prepare antisera against ChIL-18aspositive control.In this study the full-length (ChIL-18F) and mature protein of ChIL-18(ChIL-18M) cDNA wereinserted into pEGFP-N1vector, separately, and the pEGFP-ChIL-18F and pEGFP-ChIL-18M plasmidwere identified by restriction enzyme digestion and sequencing. They were transfected into CHO cellsusing Lipofectamine2000according to the manufacturer’s instruction. The cells were selected underG418. After two weeks, positive cells were cloned by limiting dilution twice and passaged usingstandard conditions. Then the cell line named as CHO-ChIL-18F and CHO-ChIL-18M were obtained.The ChIL-18F and ChIL-18M protein were confirmed by RT-PCR, indirect immunefluorescence assay(IFA) and Western blot assay. The results showed that the recombinant ChIL-18F and ChIL-18Mprotein were effectively expressed in CHO cells. This provides a useful platform for studying ChIL-18in vitro and mass production.Six-week-old female BALB/c mice were immuned by prokaryotic expressing ChIL-18protein. Afterboost immune, splenocytes and SP2/0were fused. The CHO cells which stably express ChIL-18Fprotein were fixed by acetone and methyl alcohol (v/v=1/1). The positive hybridomas were selected byIFA. After two or three subcloning, finally eight strains of monoclonal antibodies (mAbs) againstChIL-18were obtained. They were named as2G7,2G8,3E5,4E11,5D3,1E8,2C4and2D11,respectively. The heavy chains of them were IgM, light chains were κ chain. The results of Western blotassay showed that the ChIL-18mAbs only reacted with the ChIL-18recombinant protein specificlywhile did not react with the uninduce bacteria. The IL-18protein in spleens of the chicken whichinfected MDV could be detected.The established cell lines and generation of monoclonal antibodies provide useful material conditionsfor further studying on the immunological function of ChIL-18.