Objective:ToconstructamutantpEGFP-hTERTexpressionvector,toobserveitssteadyexpressionintransfectedhumanbladdercarcinomacelllineT24anditsroleinmolecularregulatorymechanismsoftelomerase,andtoprovideanewtargetgeneforbladdercancer.Methods:PCRamplificationwasperformedbyusingprimersbasedontheknowngenesequenceofhTERT.PCRproductionwasclonedintoplasmidpGEMT-TeasyandthesequenceofmutanthTERTgenewasanalyzed.ArecombinantmutanthTERTvector(pEGFP-hTERT)wasconstructedattheEcoRIandSalIsitesofthepEGFP-C1vector.AftertransfectingthefusiongeneintobladdercarcinomacelllineT24bycalciumphosphate-DNAcoprecipitation,thesteadyexpressionofGFP-hTERTfusionproteinwastestedbyfluorescentlightmicroscopy.TheproliferationchangesofbladdercarcinomacelllineT24weredetectedbylightmicroscopyandsenescencecorrelatedβ-galactosidasestaining.Results:IdentificationofpEGFP-hTERTbyenzymedigestionshowedthatmutanthTERTfragmenthadbeenclonedintoEcoRIandSalIsitesofthepEGFP-C1vector.ThesteadyexpressionofGFP-hTERTfusionproteinwaslocalizedinthenucleusoftransfectedcells.Expressionofsenescence-associatedβ-galactosidaseintransfectedcellsgraduallyincreasedwithextendedculturedtimeandcellgrowthwassuppressed.Conclusion:Themutant-typehTERTgenesuppressestheproliferationofbladdercarcinomacelllineT24bycompetitiveeffectontelomeraseactivity.ThissuggeststhathTERTgenemightbeasuitablegenetargetforbladdercancertherapy.
