Construction And Vitro Expression Of Human GM-CSF, IL-4Gene Eukaryotic Co-expression Plasmid

ObjectiveThis issue in human interleukin-4(IL-4), granulocyte macrophage colony stimulatingfactor (GM-CSF) as a model cell factor, combined to establish and optimize the humanderived cytokines in eukaryotic cells, and expression of the recombinant protein andactivity stability, provided material and theoretical reference for several cytokines in vitroCo expression. Construction of eukaryotic recombinant human GM-CSF, IL-4gene andIRES independent components series expression plasmid pGM-CSF, pIL-4and pGM-CSF-IRES-IL-4, transfected cells by immunofluorescence and connection (IFA) and enzymelinked immunosorbent assay (Elisa) to detect the expression activity, preparing for theconstruction of permanent expression cell lines, as while as human cytokine in vitroexpression and induced DC/CIK culture kit lay the foundation.Method1. Primer and plasmid design and synthesisAccording to the published GenBank human GM-CSF IL-4gene were designed, thereference sequence additional restriction site Xho I, Bgl II and Sal I, Mlu I primers andplasmids pMD15-T-CSF, pMD15-T-IL-4.2. Construction of recombinant plasmid pGM-CSF, pIL-4, pGM-CSF-IRES-IL-4PCR amplification of recombinant plasmid pMD15-T-CSF, pMD15-T-IL-4fragmentGM-CSF, IL-4; PCR agarose gel electrophoresis recovery, purification of enzyme cuttingsites respectively by double enzyme digestion, recovery of target gene GM-CSF, IL-4fragment; T4ligase respectively and the enzyme vector fragment psT-Δ SG are connected,the routine screening culture dish colony advantage resistance grow on solid, building aseparate GM-CSF, expression of recombinant plasmid of IL-4gene pGM-CSF, pIL-4; the target gene fragment IRES step by step and connection to connect to the PsT-Δ SG vector,the routine screening of culture dish colony advantage resistance grow on solid, toconstruct pGM-CSF-IRES-IL-4eukaryotic expression carrier.3. The recombinant plasmid pGM-CSF, pIL-4, pGM-CSF-IRES-IL-4transient ex-pression in CHO cellsThe recombinant plasmid pGM-CSF, pIL-4, pGM-CSF-IRES-IL-4in the positiveliposome-mediated transfection of CHO cells,24hours after detection of the expression ofthe GM-CSF, IL-4region in CHO cells.4. IFA and Elisa analysis the expressionThe protein expression of CHO cells transfected with human IFA and Elisa detectionof GM-CSF, IL-4gene.Result1. construction of the eukaryotic expression plasmid pGM-CSF, pIL-4, pGM-CSF-IRES-IL-4The eukaryotic expression plasmids of pGM-CSF, pIL-4and pGM-CSF-IRES-IL-4,respectively by colony PCR, plasmid PCR, double enzyme digestion successfullyconstructed.2. Indirect immunofluorescence assay and Elisa assay of GM-CSF, IL-4transientexpression resultsThe IFA experimental results show that under a fluorescence microscope, positivecells in more than70%. Elisa experimental results of GM-CSF, IL-4protein wassuccessfully expressed in CHO cells.Conclusion1. The success of human gene GM-CSF, IL-4subcloned into psT-Δ SG eukaryoticexpression recombinant plasmid pGM-CSF, pIL-4, pGM-CSF-IRES-IL-4.2. Successfully expressed in eukaryotic cells of human gene GM-CSF, IL-4forbuilding the of GM-CSF, IL-4section of the permanent expression cell lines and theactivation of DC/CIK cells in vitro.