简介：Ourobjectiveistosolvethelactosemalabsorptionandintoleranceofhumanbeingsbycombiningmlcro-ecologypathwithgeneticengineeringtechnique.PlasmidpMG36ewasusedtocloneandexpressaβ-galactosidasegenefromL.delbrueckiibulgaricusstrain1.1480intheLactococcuslactissubsp,cremorisMG1363andLactococcuslactissubsp.lactisIL1403.TherecombinantplasmidwaspreservedandproliferatedinEscherichiacoli(E.coli)JM109,andtransformedintoMG1363and1L1403byelectroporation.Theproteinexpressionwasstudied.(1)Thebifidobacteriumculturemedium(BBL)wassuitableforthegrowthofthestrain1.1480.(2)With13aminoacidsattheN-terminusfromthevector,β-galactosidasefusionprotein(whichretainedtheenzymeactivity)couldbesuccessfullyexpressedinE.coliJM109,MG1363andIL1403,buttheexpressionquantitywaslargerintheformerthaninthelattertwo.(3)TheSDsequencedesignedcouldbesuccessfullyrecognizedbyboththeE.coliandtheLactococcuslactis,buttheexpressionlevelofthenon-fusionβ-galac-tosidaseproteinwaslowerthanthatofthefusionproteininthesamehost.Theβ-galactosidasegeneticallyengineeredE.coliJM109isausefultooltoproducethisenzymeinvitro.Thesignalpeptideoftheusp45proteinfromtheLactococcuslactiscanbeaddedbeforethepromotersequencetopromoteβ-galactosidasesecretionfromLactococcuslactis.Thepotentialapplicationoftheβ-galactosidasegeneticallyengineeredMG1363andIL1403tocurethelactosemalabsorptionandlactoseintoleranceinbothhealthfoodandmedicineispromising。

简介：Theeffectsofvariouscartilageextracellularmatrixontheconstructionofrabbitgrowthplatecartilagetissueinvitrowerestudied.Theresultsshowthatcollagen,proteoglycanandhyaluronicacidcanpromotethegrowthofculturedchondrocytesbuttheeffectsofvariouscartilageextracellularmatrix(ECM)onchondrocytedifferentiationaredifferent.Collagencanpromotethehypertrophyofchondrocyteswhileproteoglycanandhyaluronicacidinhibitthetransitionofmaturechondrocytesintohypertrophiedchondrocytes.

简介：INTRODUCTIONThecommonconstructionmethodoftissueengineeredarticularcartilageistoisolateandculturechondrocytes,transplantthemintothedegradablescaffoldmaterials,thentorepairthedefectofarticularcartilage.Wedesignedthisexperimenttoinvestigatethefeasibilityoftissueengineeredarticularcartilageconstructedbythetechniqueofcentrifugetubeculturewithoutscaffoldmaterials.

简介：Analysisofthefrequencyofantigen-specificcytotoxicTlymphocytes(CTLs)exvivoislargelydependentontheuseofMHC/peptidetetramers.However,thelatterreagentshavenotbeenwidelyavailable,mostlikelybecauseoftheircostlyandtime-consumingproduction.InthisreportweutilizedaneconomicstrategytoconstructHLA/peptidetetramerswithrecombinantpeptide-linkedβ2microglobulin(β2m).TheHLA-A2-restricted,melanomaantigenMARTl-derivedpeptideMARTI27-35(AAGIGILTV)wasfusedtotheNterminusofhumanβ2mthrougha15-aminoacid(aa)-longlinkerbeforebeingrefoldedwiththerecombinantbiotinylatedHLA-A2heavychainectodomain.Theresulted2-component(2C)monomerwasthentetramerizedwithphycoerythin-labeledstreptavidin.Theexperimentalresultshowedthatthe2CHLA-A2/MARTI27-35monomerwasshowntobindtotheHLAclassⅠcomplex-specificmonoclonalantibodyW6/32andtheHLA-A2/MARTI27-35complex-specificsinglechainantibodyfragment(scFv)8.3,suggestingthecorrectnessofitsspecificity.Furthermore,the2CHLA-A2/MARTI27-35tetramerdetectedaspecificCD8^+TcellpopulationinHLA-A2-restrictedmelanomainfiltratinglymphocytesastheconventional3CHLA-A2/MARTI27-35tetramer.Theyieldof2CHLA-A2/MARTI27-35monomerwas2.5timesmorethanthatoftheconventional3Cmonomer.Takentogether,thesedataindicatethattheHLA-A2/MARTI27-35tetramercanbegeneratedconvenientlythroughtheuseofMARTI27-35peptide-β2mfusionproteins,whichcanfacilitatethemonitoringofHLA-A2-restricted,MARTl-specificCTLresponsesinpatientswithmelanoma.

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简介：INTRODUCTIONApproximately400millionpersonsworldwidesufferfrombladderdisease.Individualswithend-stagebladderdiseaseoftenrequirebladderreplacementorrepair.Severalbladdersubstituteshavebeenattemptedwithbothorganicmaterialsandsynthetics.

简介：IntroductionAsamemberofthebonemorphogeneticprotein(BMP)family,BMP-2playsimportantrolesnotonlyinboneregenerationandbonerepairbutalsoincellproliferation,apoptosis,differentiationandmorphogenesis.TheBMP-2remarkableabilitytostimulatenewbonegrowthresultsinthedevelopmentofanoveltherapystrategyforbonemassdefectduetoaccidentsordiseases.BecausetheBMP-2itself,inconjunctionwithasuitablematrix,issufficienttostimulategenesisofnewbone,thegeneticallyengineeredBMP-2hasgoodappliedprospects.

简介：AIM:Toconstructanon-resistantandattenuatedSalmonellatyphimurium(S.typhimurium)strainwhichexpressesconservativeregionofadhesionABofHelicobacterpylori(Hpylon)andevaluateitsimmunogenicity.METHODS:TheABgeneamplifiedbyPCRwasinsertedintotheexpressionvectorpYA248containingasdgeneandthroughtwotransformationsintroducedintothedeltaCya1deltaCrp1deltaAsdattenuatedSalmonellatyphimuriumstrain,constructingbalancedlethalattenuatedSalmonellatyphimuriumstrainsX4072(pYA248-AB).BridgedELISAmethodwasusedtomeasuretheexpressionofABantigeninsonicateandculturesupematant.AccordingtothemethoddescribedbyMeacock,stabilityoftherecombinantwasevaluated.Semi-lethalcapacitytestwasusedtoevaluatethesafetyofrecombinant.Theimmunogenicityofrecombinantwasevaluatedwithanimalexperiments.RESULTS:TheattenuatedS.typhimuriumX4072(pYA248-AB)whichexpressesABwassuccessfullyconstructed.Furthermore,bridgedELISAassayshowedthatthecontentofABinrecombinantX4072(pYA248-AB)culturesupematantwashigherthanthatwasinthalluslyticliquor.AndafterrecombinantX4072(pYA248-AB)wasculturedfor100generationswithoutselectionpressure,bheentirerecombinantbacteriaselectedrandomlycouldgrow,andtheABantigenwasdefectedpositivebyELISA.ThegrowthcurveoftherecombinantbacteriashowedthatthegrowthstatesofX4072(pYA248)andX4072(pYA248-AB)werebasicallyconsistent.ThesurvivalrateofC57BL/6wasstill100%,at30daftermicetakingX4072(pYA248-AB)1.0×l0^10cfuorally.OralimmunizationofmicewithX4072(pYA248-AB)inducedaspecificimmuneresponse.CONCLUSION:Invitrorecombinantplasmidappearstobestableandexperimentsonanimalsshowedthattherecombinantstrainsweresafeandimmunogenicinvitro,whichprovidinganewliveoralvaccinecandidateforprotectionandcareofHpyloriinfection.

简介：Objective:ToprovideahighlyefficientadenoviralvectorAd-CMV-hTGFβ1forthestudyofgenetherapyforreversionoftheintervertebraldiscdegeneration.Methods:Anewlydevelopedrecombinantadenoviralvectorconstructionsystemwasusedinthestudy.ThecDNAofhTGFβ1wasfirstsubclonedintoashuttleplasmidpShuttle-CMV.TheresultantplasmidwaslinearizedbydigestingwithrestrictionendonucleasePmeI,andsubsequentlytransformedintoE.coll.BJ5183cellswithanadenoviralbackboneplasmidpAdEasy-1.Recombinantswereselectedbykanamycinresistanceandconfirmedbyrestrictionendonucleaseanalysis.Finally,therecombinantplasmidlinearizedbyPmeIwastransfectedinto293cells.Recombinantadenovirusesweregeneratedwithin2weeks.Results:TherecombinantadenoviralplasmidswerecutbyBamHIandPacIrespectively,andthediagnosticfragmentsappearedin0.8%agaroseelectrophoresis.Theinfected293cellsshowedevidentcytopathlceffect(CPE).TheproductionsofPCRconfirmedthepresenceofrecombinantadenovirus.TheexpressionofhTGFβ1wasverifiedbyimmunohistochemicalstaining.Conclusions:ThesuccessfulgenerationoftheadenoviralvectorAd-CMV-hTGFβ1andtheconfirmationoftheinterestgeneexpressionmakeitpossiblefortheexperimentalstudyofthereversionoftheintervertebraldiscdegenerationbygenetherapy.

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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.

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简介：ObjectivesToconstructarecombinantplasmidcarryingenhancedgreenfluorescentprotein(EGFP)andhumanvascularendothelialgrowthfactor(VEGF)121geneanddetectitsexpressioninratmesenchymalstemcells(MSCs).MethodsHumanVEGF121cDNAwasamplifiedwithpolymerasechainreaction(PCR)frompCD/hVEGF121andwasinsertedintotheeukaryoticexpressionvectorpEGFPC1.AfterbeingidentifiedwithPCR,doubleenzymedigestionandDNAsequencing.TherecombinantplasmidpEGFP/hVEGF121wastransferredintoratMSCswithlipofectamine.TheexpressionofEGFP/VEGF121fusionproteinweredetectedwithfluorescencemicroscopeandimmunocytochemicalstainingrespectively.ResultsTherecombinantplasmidwasconfirmedwithPCR,doubleenzymedigestionandDNAsequencing.ThefluorescencemicroscopeandimmunocytochemicalstainingresultsshowedthattheEGFPandVEGF121proteinwereexpressedinMSCs48haftertransfection.ConclusionsTherecombinantplasmidcarryingEGFPandhumanVEGFwassuccessfullyconstructedandexpressedpositivelyinratMSCs.ItoffersapromisetoolforfurtherresearchondifferentiationofMSCsandVEGFgenetherapyforischemialcardiovasculardisease.

简介：Toinvestigatetheexistenceofthemajoroutermembranepmtein(MOMP)geneLip132in15dominantChinesestrainsof15semgroupsofLeptospirairaerrogansand2intemationalstrainsof2semgroupsofLeptospirabiflexa,andtocloneandconstructtheexpressionsystemaswellastoidentifytherecombinantpmteins,genomicDNAsfromstrainsofleptospirawerepreparedbymutinephenol-chloroformmethod,andthefragmentsoftheLipL32genewiththewholelengthfromthestrainswereamplifiedwithhighfidelityPCR.Thetargetamplificationproductswere,sequencedafterT-Acloning,andtheexpressionsystemforthegenesweretherebyconstructed,ExpressionoftherecombinantproteinswasidentifiedbyusingSDSPAGEafterinductionwithIPTGatdifferentdosages.WesternblotassayswithrabbitantiserumagainstthewholecellofTR/PatocⅠofLeptospiraandimmunizedserumwithrMOMPswereusedtodeterminetheimmunoreactivityandimmunogenicityoftherecombinantproteins.Microscopicagglutinationtestwasusedtodeterminethecross-agglutinationtitresinrabbitseraimmunizedwithrMOMPs,andthecelladherencemodelofLeptospirawasusedtoexaminetheblockingeffectsofrabbitantiseraagainsttheserMOMPs.ItwasfoundthattheLipL32genecouldbefoundinallthe17strainsofLeptospiramentionedabovewithtwodifferentgenotypes,i.e.LipL32/1andLipL32/2.AmountsofexpressionsofrMOMP1andrMOMP2afterIPTGaccountedfor40%and10%ofthetotalbacterialpmteinsrespectively.BothrMOMP1andrMOMP2couldcombinewiththerab-bitantiserumagainstleptospiralTR/PatocⅠ,andcouldinducetheproductionofagglutinationantibodiestothese17strainsofLeptospirawith1:2to1:64MATtitres.Therabbitanti-rMOMP1andanti-MOMP2antibodiesat1:2to1:16dilutionscouldefficientlyblockadherenceofLeptospira.ItconcludesthatalltheLeptospiratestedinthepresentstudypossessLipL32/1orLipL32/2genes,andtheconstructedexpressionsystemcanexpresstherMOMP1andrMOMP2.

简介：AIM:RecombinedplasmidpETNF-P16wasconstructedtoinvestigateitsexpressionpropertiesinesophagealsquamouscarcinomacelllineEC9706inducedbyX-rayirradiationandthefeasibilityofgene-radiotherapyforesophagealcarcinoma.METHODS:RecombinedplasmidpETNF-P16wasconstructedandtransfectedintoEC9706cellswithlipofectamine.ELISA,Westernblot,andimmunocytochemistrywereperformedtodeterminetheexpressionpropertiesofpETNF-P16inEC9706aftertransfectioninducedbyX-rayirradiation.RESULTS:EukaryoticexpressionvectorpETNF-P16wassuccessfullyconstructedandtransfectedintoEC9706cells.TNFαexpressionsweresignificantlyincreasedinthetransfectedcellsafterdifferentdosesofX-rayirradiationthaninthoseafter0Gyirradiation(1192.330-2026.518pg/mL,P<0.05-0.01),andtheTNFαexpressionsandP16weresignificantlyhigher6-48hafter2GyX-rayirradiation(358.963-585.571pg/mL,P<0.05-0.001).NoP16expressionwasdetectedinnormalEC9706cells.However,therewasstrongexpressioninthetransfectedandirradiationgroups.CONCLUSION:X-rayirradiationinductioncouldsignificantlyenhanceTNFαandP16expressioninEC9706cellstransfectedwithpETNF-P16plasmid.Theseresultsmayprovideimportantexperimentaldataandtherapeuticpotentialforgene-radiotherapyofesophagealcarcinoma.

简介：ThepresentstudyisaimedatstudyingthegeneforTIMP-3,amammaliantissueinhibitor,byconstructingarecombinanteukaryoticcellvectorforgenetherapyinhumanbreastcancer.WeobtainedtheTIMP-3genefromthehumanplacentbyRT-PCR.TIMP-3genewassubclonedintopcDNA3.1vetorfrompMD18TvectorbymeansofgenecloningtoconstructpcDNA3.1recombinantvector.HumanbreastcancercelllineMDA-MB-453wastransfectedwithpcDNA3.1-TIMP3recombinantvectorusinglipofectaminereagent.ThentheexpressionofTIMP-3andtheeffectonthemetastasisofMDA-MB-453wereexamined.ThecorrectconstructionofpcDNA-TIMP3wasidentifiedbymeansofrestrictionenzymeanalysis,PCRamplicationandnucleotidesequencing.WesternblottingshowedthatthetransfectedcellswereabletoexpressTIMP-3,indicatingthatourconstructionofthepcDNA-TIMP3eukaryoticexpressionvectorwasconstructedsuccessfully.OurexperimentsfurtherindicatedthatthepotentialofmetastasiswassignificantlyreducedforthetransfectedcelllineMDA-MB-453.

简介：ToconstructandexpressthefusionproteinStx2B-IntiminC300ofEHEC0157:H7,andtofurtherinvestigateitsimmunoprophyiacticpotential,thegeneofStx2B(stx2b)fromEHEC0157:H7chromosomewasclonedintopMD18-Tvector.Thereafter,theamplifiedgenewasclonedintoprokaryoticexpressionplasmidpET-28a(+)-eaeC300,whichwasconstructedpreviously.TherecombinantpasmidpET-28a(+)-stx2b-eaeC300wastransformedintoE.coliBL21(DE3).Afterinducement,theproteinStx2B-IntiminC300wassuccessfullyexpressedandanalyzedwithsodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE),WesternblottingandN-terminalaminoacidresidualsequencing.Toevaluateitsimmunoprophyiacticpotential,itwasprimarilypurifiedbyion-exchangechromatographyandinjectedinto30BALB/cmicewithAl(OH)3inthesubscapularregion.Tendaysafterthelastboostervaccination,20micewereattackedwithEHEC0157:H7lysateandtheprotectiveefficacywasobserved.Inthepresentstudy,thegeneofStx2B-IntiminC300wassuccessfullyclonedintopET-28a(+)vector.TheresultsofSDS-PAGEandWesternblottingassayshowedthatthefusionproteinwassuccessfullyexpressedintheinclusionbodyform,accountingfor25%oftotalexpressionproducts,anditsmolecularweightwasabout43kDa.TheresultoftheN-terminalaminoacidresidualsequencingshowedthatitwasidenticaltothatofthemoleculardesigned.Thepuritywasabout75%afterprimarypurification.AnimaltestsrevealedthatthefusionproteinStx2B-IntiminC300haselicitedhightiterofprotectiveantibodyrelatively.TheseresultsdemonstratethatthefusionproteinStx2B-IntiminC300issuccessfullyexpressedinprokaryoticexpressionsystemandshowscertainimmunoprophyiacticpotential.

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