Construction of eukaryotic vector pEGFP-N1-IL-17 and screening of stably transfected pEGFP-N1-IL-17-U87MG cell

IL-17 has been demonstrated to promote tumor growth via certain pathways.In current studies IL-17 has been found to be highly expressed in glioma.This research aims to construct the eukaryotic vector pEGFP-N1-IL-17 and makes it to be stably expressed in glioma cell line U87MG in order to provide the basis for studying its function in glioma progress.PBMC was collected from an idiopathic thrombocytopenic purpura(ITP) patient and the RNA was extracted.IL-17 cDNA was synthesized and cloned into PMD 19-T and pEGFP-N1 plasmid by SaIⅠ and BamH enzymatic digestion.The clones were identified by enzymatic digestion and sequencing.The correct clone was amplified and plasmid DNA was then transfected into glioma cell line U87MG.pEGFP-N1-IL-17-U87MG cells that stably expressed IL-17 were obtained by limited dilution by G418 added.Correct clone was identified by fluorescence,qRT-PCR and ELISA.The results showed that IL-17 cDNA、PMDR 19-T-IL-17,and pEGFP-N1-IL-17 were successfully obtained.And pEGFP-N1-IL-17-U87MG cells that stably express IL-17 were identified through fluorescence,qRT-PCR and ELISA.MCP-1 mRNA was down regulated after U87MG cell was transfected by IL-17.Therefore the eukaryotic expression vector pEGFP-N1-IL-17 has been successfully constructed and stably transfected into U87MG cell which could be used for study on IL-17's function in glioma tumorigenesis.