Research On The Construction Of The Eukaryotic Expression Vector PEGFPN1-TRAIL

Purpose:To obtain the eukaryotic expression vector pEGFPN1-TRAIL,which contain the TRAIL114-281AA gene fragment.Transfect the plasmidpEGFPN1-TRAIL and pEGFPN1,respectively,into the human hepatocellularcarcinoma cell line HepG2cells.primary study on the expression of TRAILgene in HepG2cells and it is good for the further researches in vivo and invitro.Methods:The TRAIL114-281AA coding region was amplified fromFL15116plasmid by PCR,then the TRAIL gene fragments were purified byDNA Gel Extraction Kit and cloned into T expression vectorPMD18-T.Transform the competent cells named DH5,then pick themonoclonal colony to amplify.PMD18-T-TRAIL plasmid was extracted foridentification.After the identification of PCR,restriction enzyme digestion andsequencing by TAKARA company.TRAIL gene from PMD18-T-TRAIL byrestriction enzyme was cloned into the vector pEGFPN1.The recombinantplasmid was named pEGFPN1-TRAIL.It can be tested by the restrictionenzyme.Transform it into human hepatocellular carcinoma cell line HepG2cells by lipofectamine2000transfection reagent.TRAIL expression in thepEGFPN1-TRAIL transfected HepG2was verified by RT-PCR.Results:The TRAIL114-281AA gene fragments was amplified by PCRsuccessfully.The production after the connection between PMD18-T plasmidand TRAIL gene was identified by the method of PCR,enzyme-cleavagereaction. Target genes was obtained,which was about500bp.There was nomutation after the sequencing identification.The TRAIL gene fragment was subcloned into the eukaryotic expression plasmid pEGFPN1.After therecombinant plasmid pEGFPN1-TRAIL was transfected into the HepG2cells,the TRAIL gene could be examed expressed under the fluorescencemicroscope and over expressed in the HepG2cells using the RT-PCR method.Conclusion:The eukaryotic expression vector pEGFPN1-TRAIL wasconstructed successfully.After the recombinant pEGFPN1-TRAIL plasmidand the plasmid pEGFPN1was transfected into HepG2cells,respectively.theTRAIL gene of the experimental group was tested over expressed in theHepG2cells by RT-PCR.This research could be good for the further studyabout protein and animal study.The TRAIL gene may serve as a noveltherapeutic strategies for HCC in the future.