Construction Of Interleukin-18-PE38 Fusion Gene Eukaryotic Expression Vector And The Primary Study Of It Against Disorders

Objectives:To construct eukaryotic expression vector of recombinate immunotoxin IL-18-PE38 fusion gene, and evaluate therapeutic efficacy of this IL18-PE38 fusion protein for leukemia and rheymetoid arthritis which overexpress IL-18 receptor.Methods and Results:(1) Interleukin-18-PE38 fusion gene from plasmid PRKL459k-IL-18-PE38 was cleaved by restriction enzyme digestion, it was linked into eukaryotic secretion vector PsecTag2B and was transformed into competent bacteria, the clones were selected through antibody culture medium. The positive clones were confirmed by restrictive enzyme (EcoRI) digestion assay and DNA sequencing. Restriction endonuclease digestion analysis revealed that the length of the interleukin-18-PE38 recombinant plasmid was 6.8kb. DNA sequencing showed that IL-18 and PE38 sequence matched with reported sequence in the genebank.(2) IL-18-PE38 plasmid was transfected into NIH3T3 cells by liposome protocol, control group was established with vacant vector, and the transient expression was identified by fluorescence immunocytochemical assay. Photograph of fluorescence immunocytochemical method showed thatfluorescence intensity representing IL-18-PE38 expression of the experimental group which was transfected with IL-18-PE38 recombinant plasmid is strong, while the fluorescence intensity of the control group which was transfected vacant plasmid is weak in NIH3T3 cells. Feed NIH3T3 cells with Zeocin selective medium until Zeocin-resistant cell foci are identified, fifty days after feeding NIH3T3 cells with Zeocin selective medium, cells transfected no plasmid were completely died, whereas cells in the control group and experiment group were formed into clones. The expression of fusion protein in the cell culture fluid was certified through spot ELLSA and Western-bloting. Incubate leukemia L615 cells with transfected cell culture fluid, cell growth was measured through MTT assay, and cell apoptosis was detected through flow cytometry. MTT assay showed higher L615 death rate in the experiment group than control group. Flow cytometry analysis showed distinct apoptotic peak in the experiment group. Immuocytochemistry photograph of AO/EB fluorescence double staining demonstrated distinct apoptotic cells staining feature in the experimental group.(3) Primary chrondrocyte culture of mouse was constructed. It was identified by collagen II immunocytochemical assay. Collagen II immunocytochemical staining of primary cultured mouse chrodrocyte showed the positive staining in cytoplasm. IL-18-PE38 plasmid was transfected into chondrocyte by liposome protocol, control group was established with vacant vector,and the transient expression was identified by fluorescence immunocytochemical assay. Photograph of fluorescence immunocytochemical assay showed that fluorescence intensity representing IL-18-PE38 expression of the experimental group which was transfected with IL-18-PE38 recombinant plasmidis strong, while the fluorescence intensity of the control group which was transfectedcacant plasmid is weak in chondrocyte . Conclusions:(1) The eukaryotic expression vector PsecTag2B-IL-18-PE38 was established successfully.(2) Recombinant immnotoxin IL-18-PE38 fusion protein was expressed and secreted by NIH3T3 cells which were transfected IL-18-PE38 plasmid, and the fusion toxin can induce L615 cells apoptosis. Which suggested that the recombinant immunotoxin can be prospecting to treat leukemia.(3) Mouse primary chrondrocyte line was constructed. The fusion gene was expressed in mouse chrodrocyte successfully. Which suggested that the recombinant immunotoxin can be prospecting to treat rheumatoid arthritis.