Study On The Biological Activities Assay Of Recombined Chicken Interleukin-18 And Its Effect On Chicken Immunity

Interleukin 18(IL-18) has multiple biological activities including induction of IFN-γfrom NK cells and antigen- or mitogen-stimulated Th1 cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NK cell cytotoxicity. These on human beings and animals were identified by many documents recently. Chickens IL-18 was constructed to recombinant chicken IL-18 genes in our previous study in 2003. The bioactivities of expressed product of ChIL-18 in E.coli would be assayed and the methods of purification and renaturation will be assessed and the effect of it on the immune in chickens will be study as following.1 Optimal Conditions in the MTT Assays to Detect the Cytotoxicity of NK CellsThe cytotoxicity of NK cells isolated from blood and spleen of different-day-chickens as effect cells to target cells, MDCC-MSB1 and MSBCU147, were detected by MTT assays and evalue the ratio of effect cells and target cells and the time of incubation. The result indicated MSB1 and MSBCU147 could be better target cells in the assay of NK cells cytotoxicity and the optimal conditions were 20:1~50:1 as the ratio of effect cell and target cells and 8~16h of incubation and the 20%SDS-50%DMF mixture as lytsis to MTT product. This will be help to analyze the cytotoxicity of NK cells in chickens.2 Biological Activities Assay of Purified Expressed Product of Recombined Chicken Interleukin-18 Gene in E.coliPositive vector containing chicken IL-18 gene was expressed in E.coli, then the expressed product was purified by affinity chromatography, the activity of IFN-γinduced by rChIL-18 and of rChIL-18 antivirus were detected with CEF-VSV system, 3H-thymidine (3H-TdR) incorporation assay and MTT colorimetric assay were used to detect T lymphocytes proliferation response to rChIL-18 and the effect of rChIL-18 on cytotoxicity of NK cells, respectively. Results, recombinant protein with a relative molecular weight of 44 KD (including 26KD GST) was expressed in E.coli, which could induce splenocytes to produce IFN-γwith high activity, but could not directly inhibit the growth of VSV in CEF. It was proved that purified protein could stimulate T lymphocytes proliferation and increase NK cytotoxicity at certain concentrations. It can be concluded that chicken IL-18 protein...