| Interleukin-18 (IL-18) was first purified by Okamura in 1995 from thelivers of mice treated with the bacterium P. acnes and subsequentlychallenged with lipopolysaccharide (LPS) to induce toxic shock。Because itcan induce the producing of IFN-γ by the Thl cell,it was first calledinterferon-γ一inducing factor,then it is named interleukin-18 in 1996。AcDNA of humanIL-18 cloned by cross-hybridization from human liver cDNAlibraries was found to contain a single open reading frame encoding a193-amino-acid proIL-18, ProIL-18 has no N-glyco site and conventionalsignal peptide。It has been shown that the biological inactive proIL-18 iscleaved with interleukin-1β(IL-1β)-converting enzyme (ICE) at the authenticprocessing site, Asp-X, to generate the mature active form of IL-18.。HumanIL-18 is mainly produced by peripheral blood mononuclear cells (PBMC)and macrophages, IL-18 enhances cytotocity of NK and proliferation of Tcells, It stimulates Thl cells to produce IL-2 and IFN-γ,This effect isaugmented by IL-12 in a synergistic manner。 In addition, IL-18 canaugments the production of GM-CSF and decreases IL-10 production but noteffect on IL-4 secretion by PBMC。Currently, the former clinical research applications of IL-18 are active,the application areas including anti-tumor, anti-infection and the treatment oftransplant material-host reaction (GVHD), in which the main anti-infectionincluding anti-virus, anti tubercle bacillus, anti-fungal and anti-parasiticinfections, such as the placenta, have greater prospects for clinicalapplications. People in recent years launched a domestic IL-18 (hIL-18)expressed research cloning, but the volume is not very good expression, andfor the integration of multi-expression is unsuitable for hIL-18 drugdevelopment and clinical applications. Therefore, we use synthetic methodsredesign IL-18 genes, a type of expression plasmids by non-fusion, and wasexpressed at a high level in E.coli.The main papers for the following aspects :1) From the use of codon, adjustment G+C% content and distribution, thenumber of mRNA stability perspective on human Interleukin-18 genes tomake an entirely new design, and synthetic human Interleukin-18 genes.2) New synthetic gene was constructed into different pPL450 vector,pBV220 vector,pET20b(+) vector, and then transformed to engineeringbacteria,expressed the recombinant hIL-18 protein. To be the product ofanalysis by more pBV220 vector for the best expression of the vector,reflecting induce simple methods to induce expression of the largest, low-cost,easy to operate, and other advantages. At the same time compared to the samenew gene expression in the vector pBV220 volume from 14.384% to29.931%, and then the basis for inducing time was further optimization..3) RhIL-18 protein sample separation and purity study, the rhIL-18 nonfusionprotein was express as insoluble aggregates system ,in order to get thepurified IL-18,the products were by the inclusion bodies extraction, theinclusion bodies washing, denaturalization,ion exchange and SephacrylS-100 purfication steps, available for more than 95% purity proteinexpression, we set up a laboratory-scale purification platform.4) In addition, the recombinant hIL-18 was constructed into pRSET (his)vector, and purfied the IL-18, immunization female rabbit got IL-18multi-clone antibodies to provide IL-18 Atellin preparation for the later basis.Based on the above, the completion of this paper, synthetic IL-18expressed significantly enhanced laboratory and the establishment of purfityplatform for the coming applications hIL-18 gene therapy and protein therapyown immunological diseases provided favorable conditions. |
PDF Full Text Request