Expression And Purification Of Chicken Mature Interleukinl8and Preparation And Identification Of Its Monoclonal Antibodies

Interleukin18can significantly induce Th1cells and NK cells to produce IFN-γ, also caninduce the production of IL-2、GM-CSF and TNF-α and other multiple cytokines. IL-18notonly has anti-viral, anti-tumor activity, can also be used as immune adjuvant to strengthen andimprove the immune response. The co-expression of ChIL-18gene and virus protectiveantigen gene can strengthen and improve the immune response. Monitoring of IL-18level incertain diseases can dynamically observe and understand the disease, prognosis, and the lawof the body’s immune response. It is necessary to use anti-IL-18monoclonal antibody todevelop the detection method. So far, the products of human, mouse, monkey, porcine, bovine,goat IL-18and the monoclonal or polyclonal antibody reagents against them have beendeveloped, as well as the relative double-antibody ELISA kits. However, the function andrelative studies of Chicken IL-18(ChIL-18) is still poorly understood. Even no any productsof anti-ChIL-18monoclonal come out yet.In this study, the McAbs anti-mature Chicken interleukin-18(mChIL-18) were preparedand studied. The gene of mChIL-18was amplified and cloned into a pET-28a(+) vector. Byidentification and sequencing, the recombinant plasmid pET-28a-ChIL-18was constructed.The recombinant plasmid pET-28a-ChIL-18was transformed into Rosetta (DE3) competentcells. Using IPTG to induce and obtaining the expression of the recombinant proteins. Usingthe feature of the His tags in the vector, the recombinant ChIL-18proteins were purified byNi-NTA Resin affinity chromatography. The purified proteins were used for the immunity ofthe mice, screening and identification of the McAbs.The BALB/c mice were immunized with purified ChIL-18proteins. And, the indirectELISA detection was constructed for detecting level of mice’ antibodies and screeninghybridoma cells. The optimum working conditions for the indirect ELISA are as follows:optimal concentration of mChIL-18for coating was4μg/mL; the dilution of serum sample was1:104; the optimal coating condition of mChIL-18was incubated at4℃over night; thebest dilution of HRP-labeled goat anti-mouse IgG was1:4000; the best incubation period andtemperature for the serum sample and HRP-labeled goat anti-mouse IgG was37℃for1.5hand37℃for1h, respectively. The titers of mice were up to1:106before the fusion at thesatisfaction of the conditions of the fusion. The splenocytes were fused with SP2/0myelomacells under the effect of PEG. The fusion cells were detected by indirect ELISA. The positivehybridoma cells were sub-cloned three times or four times by limited dilution methods toobtain the hybridoma cells secreting antibodies stably. Finally, the two McAbs (1G9and2E6)were obtained. The ascites fluids were harvested.The identification of isotype of McAbs showed that the two McAbs were IgM isotype. Theascites titers of1G9and2E6were1:5.12×105and1:3.2×104, respectively. ELISA additity testshowed that the binding of two McAbs,2E6and1G9, was additive and both bound to thedistinct epitopes. Western blot showed that two McAbs,2E6and1G9, could react withrecombinant mChIL-18proteins specifically and could not react with Newcastle Disease NPproteins and Avian Metapneumovirus proteins which were expressed in Rosetta (DE3) withpET-28a(+). The McAbs could be used to detect the expression of recombinant eukaryoticplasmid pcDNA3.1-ChIL-18transfected into the293FT cells specifically by IFA. IFAdisplayed intense green fluorescence.