简介：

简介：Objective:ToconstructtherecombinantplasmidcontainingGlycerophosphodiesterphosphodiesterase(Gpd)genefromTreponemapallidumandtransfectitintoHelacellstoexpresstheencodedoutermembraneprotein.Methods:TheGpdgenewasamplifiedfromthegenomicDNAofT.pallidumbypolymerasechainreaction(PCR)andinsertedintocloningvectorpUCm-T.TheinsertedGpdgenewassubclonedintotheappropriatesiteofpcDNA3.1(+)vector.Afteridentificationbysequencingandrestrictiveenzymesdigestion,therecombinantplasmidwastransfectedintoHelacellsusingliposomes.TheexpressedproteinwasidentifiedbyimmunocytochemistryandWesternblot.Results:ThetargetGpdgenesegmentwasapproximately1059bp.TheDNAsequenceoftheGpdgenecontainedinthepcDNA3.1(+)vectorwasconsistentwiththepublishednucleotidesequence.ThehomologyofthenucleotideandputativeaminoacidsequencesoftheGpdgenebetweenT.pallidumsubsp.pallidumNicholsandvariouspathogenictreponemalstrainsrangedfrom98%to100%.ImmunocytochemistryandWesternblotanalysisshowedthattheconstructedGpd-pcDNA3.1(+)vectorexpressedafusionproteinwithacalculatedmolecularmassof41KDainHelacellsandthattheexpressedproteinreactedwiththeserafromsyphilispatients.Conclusion:ThesuccessfulconstructionandexpressionoftheeukaryoticexpressionplasmidoftheGpdgenefromT.pallidumprovideapromisingtooltofurtherstudythebiologicalactivityofT.pallidumanddevelopaDNAvaccineforsyphilis.