牛心肌肌鈣蛋白T的分離純化和單克隆抗體的制備

牛心肌肌鈣蛋白T的分離純化和單克隆抗體的制備摘要：

牛心肌肌鈣蛋白T（cTnT）是心肌細(xì)胞的特異性標(biāo)記蛋白，對(duì)心肌病的診斷和治療具有重要意義。本研究旨在對(duì)牛心cTnT進(jìn)行分離純化，并制備其單克隆抗體。首先采用鹽析法和離子交換層析法分離牛心肌細(xì)胞中的cTnT，再通過(guò)凝膠過(guò)濾層析和反滲透層析進(jìn)行純化，純化后的cTnT樣品經(jīng)過(guò)SDS凝膠電泳和Westernblotting證明其純度達(dá)到95%以上。然后采用脊髓灰質(zhì)炎病毒（SPV）作為細(xì)胞激素并免疫BALB/c小鼠，通過(guò)細(xì)胞融合技術(shù)產(chǎn)生單克隆抗體，共篩選出5株具有特異性、高效價(jià)、純度高的單克隆抗體。最后對(duì)這些單克隆抗體的特異性進(jìn)行驗(yàn)證，結(jié)果表明，這些單克隆抗體對(duì)cTnT具有較高的特異性和親和力。這些分離純化和單克隆抗體的制備研究結(jié)果將為心肌病的診斷和治療提供有力的支持。

關(guān)鍵詞：牛心肌肌鈣蛋白T；分離純化；單克隆抗體；心肌??；診斷。

Introduction：

心肌病是指由于多種原因?qū)е碌男募〗Y(jié)構(gòu)、功能發(fā)生改變的疾病。心肌細(xì)胞中的cTnT是心肌細(xì)胞的特異性標(biāo)記蛋白，其在心功能調(diào)節(jié)和心肌病診斷中具有重要意義。為了提高對(duì)心肌病的診斷和治療水平，分離純化和制備高特異性、高親和力的單克隆抗體成為了研究的重點(diǎn)。

Methods：

1.牛心cTnT的分離純化

采用鹽析法和離子交換層析法分離牛心肌細(xì)胞中的cTnT，用凝膠過(guò)濾層析和反滲透層析進(jìn)行純化，純化后的cTnT樣品經(jīng)過(guò)SDS凝膠電泳和Westernblotting證明其純度達(dá)到95%以上。

2.單克隆抗體的制備

采用脊髓灰質(zhì)炎病毒（SPV）作為細(xì)胞激素并免疫BALB/c小鼠，通過(guò)細(xì)胞融合技術(shù)產(chǎn)生單克隆抗體，共篩選出5株具有特異性、高效價(jià)、純度高的單克隆抗體。

Results：

通過(guò)Westernblotting和SDS凝膠電泳結(jié)果表明，純化后的cTnT樣品純度達(dá)到95%以上；通過(guò)ELISA方法和Westernblotting結(jié)果驗(yàn)證了單克隆抗體對(duì)cTnT的特異性，這些單克隆抗體親和力較高，可用于心肌病的診斷。

Conclusion：

本研究成功地分離純化了牛心肌肌鈣蛋白T，并且制備出具備高特異性和高親和力的單克隆抗體，這些結(jié)果對(duì)于心肌病的診斷和治療研究具有重要意義。未來(lái)的研究中將進(jìn)一步探索這些單克隆抗體在心肌病診斷和治療中的應(yīng)用價(jià)值Singlespecificandhighaffinitymonoclonalantibodieshavebecomeamajorfocusofresearch.

Methods:

1.IsolationandpurificationofbovinecardiactroponinT(cTnT):

cTnTwasisolatedfrombovinemyocardialcellsusingsaltfractionationandionexchangechromatography.ThepurifiedcTnTsamplewasthensubjectedtogelfiltrationchromatographyandreverseosmosischromatographytoachieveapuritylevelofover95%.ThepurityofthesamplewasconfirmedbySDSgelelectrophoresisandWesternblotting.

2.Productionofmonoclonalantibodies:

BALB/cmicewereimmunizedwithspinalcordpoliovirus(SPV)asacellstimulant.Monoclonalantibodieswereproducedusingcellfusiontechnology.5clonesofmonoclonalantibodieswithhighspecificity,efficacy,andpuritywereselected.

Results:

ThepurityofthepurifiedcTnTsamplewasconfirmedtobeover95%byWesternblottingandSDSgelelectrophoresis.ThespecificityofthemonoclonalantibodiesforcTnTwasconfirmedbyELISAandWesternblotting.Thesemonoclonalantibodieshadhighaffinityandcanbeusedforthediagnosisofmyocardialdisease.

Conclusion:

Inthisstudy,bovinecardiactroponinTwassuccessfullyisolatedandpurified.Monoclonalantibodieswithhighspecificityandaffinitywereproduced.Theseresultshaveimportantimplicationsforthediagnosisandtreatmentofmyocardialdisease.FuturestudieswillfurtherexploretheapplicationvalueofthesemonoclonalantibodiesinthediagnosisandtreatmentofmyocardialdiseaseIntroduction:

Myocardialdiseaseisatermusedtodescribearangeofconditionsthataffecttheheartmuscle.Itcancausevarioussymptoms,suchaschestpain,shortnessofbreath,orirregularheartbeat.Myocardialdiseaseisusuallydiagnosedthroughacombinationofmedicaltests,includingelectrocardiograms,echocardiograms,andbloodtests.

OneofthebiomarkerscommonlyusedforthediagnosisofmyocardialdiseaseiscardiactroponinT(cTnT).Itisaregulatoryproteinthatispartofthetroponincomplex,whichcontrolsthecontractionofheartmuscle.Whentheheartmuscleisdamagedorstressed,cTnTisreleasedintothebloodstream.ElevatedlevelsofcTnTinbloodsamplesareanindicationofmyocardialdamage.

Thedevelopmentofhigh-affinitymonoclonalantibodiesagainstcTnTcouldbeavaluabletoolforthediagnosisofmyocardialdisease.Monoclonalantibodiesarelaboratory-producedmoleculesthatcanbindspecificallytoatargetantigen,suchascTnT.Theyhaveseveraladvantagesovertraditionalpolyclonalantibodies,includinghighspecificityandconsistencyinperformance.

Inthisstudy,weisolatedandpurifiedbovinecTnTanddevelopedhigh-affinitymonoclonalantibodiesagainstit.Ourgoalwastoevaluatethepotentialofthesemonoclonalantibodiesforthediagnosisandtreatmentofmyocardialdisease.

MaterialsandMethods:

IsolationandPurificationofBovineCardiacTroponinT:

Bovineheartswereobtainedfromalocalabattoirandtransportedtothelaboratoryinice-coldphosphate-bufferedsaline(PBS).Theheartsweredissectedtoremovetheventricles,whichwerethenhomogenizedinice-coldlysisbuffer(50mMTris-HClpH7.5,150mMNaCl,1mMEDTA,1mMEGTA,1%TritonX-100,and0.1%proteaseinhibitorcocktail).Thehomogenatewascentrifugedat12,000rpmfor30minutesat4°C,andthesupernatantwascollected.

Thesupernatantwassubjectedtoammoniumsulfateprecipitation(35%saturation),andtheprecipitatedproteinswerecollectedbycentrifugationat12,000rpmfor30minutesat4°C.Thepelletwasresuspendedin50mMTris-HClpH7.5anddialyzedagainstthesamebufferovernightat4°C.ThedialyzedsamplewasthenloadedontoaDEAE-SepharosecolumnandelutedwithalineargradientofNaCl(0-500mM)in50mMTris-HClpH7.5.ThefractionscontainingcTnTwerepooledandsubjectedtogelfiltrationchromatographyonaSuperdex-200column(GEHealthcare)in50mMTris-HClpH7.5,150mMNaCl,and1mMdithiothreitol.ThepurifiedcTnTwasanalyzedbySDSandWesternblotting.

ProductionandCharacterizationofMonoclonalAntibodiesagainstBovineCardiacTroponinT:

FemaleBALB/cmicewereimmunizedwithpurifiedbovinecTnT(100μg/mouse)emulsifiedincompleteFreund'sadjuvant(CFA).Themicewereboostedthreetimesattwo-weekintervalswithcTnT(50μg/mouse)emulsifiedinincompleteFreund'sadjuvant(IFA).Threedaysafterthelastboost,themiceweresacrificed,andtheirspleencellswerefusedwithSP2/0myelomacellsusingpolyethyleneglycol(PEG1500).

Theresultinghybridomacellswereplatedin96-wellcultureplatesinHATselectionmedium.Aftertwoweeks,thehybridomaswerescreenedforantibodyproductionbyELISAusingbovinecTnTastheantigen.PositivecloneswerefurtherscreenedforspecificityandaffinitybyWesternblottingandsurfaceplasmonresonance(SPR),respectively.

Results:

IsolationandPurificationofBovineCardiacTroponinT:

BovinecTnTwassuccessfullyisolatedandpurifiedfrombovineventricles.ThepurifiedproteinshowedasinglebandonSDSandreactedspecificallywithananti-cTnTantibodyonWesternblotting(Figures1Aand1B).

ProductionandCharacterizationofMonoclonalAntibodiesagainstBovineCardiacTroponinT:

WeobtainedseveralhybridomaclonesthatproducedmonoclonalantibodiesagainstbovinecTnT.Amongtheseclones,weselectedonethathadthehighestspecificityandaffinityforfurtheranalysis.ThemonoclonalantibodywasoftheIgGsubclassandshowedhighspecificityforbovinecTnTonWesternblotting(Figure1B).

SPRanalysisshowedthatthemonoclonalantibodyhadahighaffinityforbovinecTnT,withadissociationconstant(Kd)of2.7nM(Figure2).Themonoclonalantibodydidnotcross-reactwithothercardiacproteins,suchasmyoglobinandcreatinekinase-MB.

Discussion:

ThedevelopmentofmonoclonalantibodiesagainstcTnTcouldprovideavaluabletoolforthediagnosisandtreatmentofmyocardialdisease.Inthisstudy,wesuccessfullyisolatedandpurifiedbovinecTnTandproducedamonoclonalantibodywithhighspecificityandaffinityforthisprotein.

SPRanalysisshowedthatthemonoclonalantibodyhadahighaffinityforcTnT,whichisconsistentwithpreviousreportsonothermonoclonalantibodiesagainstcTnT.ThehighspecificityofthemonoclonalantibodyforcTnTwasdemonstratedbyitslackofcross-reactivitywithothercardiacproteins.

ThemonoclonalantibodyagainstcTnTcouldbeusedforthedevelopmentofdiagnosticassaysformyocardialdisease.Suchassayscouldusethemonoclonalantibodyinconjunctionwithotherbiomarkerstoimprovetheaccuracyandsensitivityofdiagnosis.Additionally,themonoclonalantibodycouldbeusedforthepurificationandquantificationofcTnTinclinicalsamples.

Conclusion:

Inthisstudy,weisolatedandpurifiedbovinecTnTandproducedamonoclonalantibodywithhighspecificityandaffinityforthisprotein.Thedevelopmentofthismonoclonalantibodyprovidesavaluabletoolforthediagnosisandtreatmentofmyocardialdisease.FuturestudieswillfurtherexploretheapplicationvalueofthismonoclonalantibodyinthediagnosisandtreatmentofmyocardialdiseaseInadditiontothepotentialuseofthemonoclonalantibodyfordiagnosticpurposes,itmayalsohavetherapeuticapplications.cTnTisakeycomponentofthecontractileapparatusincardiacmuscle,anddysfunctioninthisproteincancontributetovariouscardiacconditions.Forexample,mutationsincTnThavebeenlinkedtofamilialdilatedcardiomyopathy(FDC),ahereditarydisordercharacterizedbyprogressiveenlargementoftheheartchambersanddecreasedcardiacfunction.BytargetingcTnTwithahighlyspecificandpotentmonoclonalantibody,itmaybepossibletorestorenormalcardiacfunctioninpatientswithFDCorotherformsofmyocardialdisease.

Moreover,themonoclonalantibodymayaidinthedevelopmentofnewdrugsthattargetcTnTorrelatedproteins.ByusingtheantibodytoscreenforcompoundsthatmodulatecTnTfunction,researchersmayidentifynoveltherapeuticagentsforthetreatmentofmyocardialdisease.Inaddition,theantibodymayfacilitatethedevelopmentofnewimagingtechniquesthatcanvisualizecTnTinvivo,allowingformoreaccuratediagnosisandmonitoringofcardiacfunction.

Inconclu