Cloning, expression and function of the extracellular fragment of human TRAIL gene-中国期刊网

摘要 ToobtaintherecombinantsolubleproteinoftheextracellularfragmentofhumanTRAILgeneandtoidentifyitsfunctionpreliminarily,thisgenefragmentwasamplifiedfromperipheralbloodmononu-clearcells(PBMC)byRT-PCRandclonedintovectorpGEM-T-Easyforsequenceanalysis.Theexpres-sionvectorpET-30a/TRAILwasthenconstructedbyDNArecombinationmethodwithaHis-taggeneatthefrontoftheTRAILfragment,andtherecombinantproteinwasexpressedinE.coliBL21(DE3).Meanwhile,theexpressedtargetproteinwaspurifiedwithNi-NTAchromatographycolumnandidentifiedbySDS-PAGEandWesternblotting.TheproliferationinhibitionactivityofTRAIL-HiswasdetectedbyMTTassay.PIstainingandWright-GiemsastainingwereusedtodetectthepresenceoftheTRAIL-in-ducedcellapoptosis.ItwasdemonstratedthatthetargetproteinexpressedinE.coliBL21showedthesamerelativemolecularmassasthattheproteinexpectedandcouldberecognizedbyboththeanti-TRAILpolyclonalantibodyandanti-Hismonoclonalantibody.Inaddition,thisproteincouldalsoinhibitprolif-erationofhumanlymphomacelllineJurkatcellsorinduceapoptosisofthiscellline.ItisapparentthatarecombinantsolubleTRAILproteinwithbiologicalactivityisobtainedandthisprospectivestudycanlaysolidfoundationforfurtherresearchonthebiologicalactivityandapplicationintheanti-tumortherapy.

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Cloning, expression and function of the extracellular fragment of human TRAIL gene

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Cloning, expression and function of the extracellular fragment of human TRAIL gene

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