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.

简介：Objective:Toclone,sequenceandexpresstheprimateβ-chemokineRANTESgenes,hRANTESfromH.sapiensandmRANTESfromM.Mulatta,inordertoexplorethepossibilityofAIDSgenetherapy.Methods:hRANTESandmRANTESwereamplifiedbyreversetranscription-polymerasechainreaction(RT-PCR)fromRNAsextractedfromphytoagglutinin(PHA)-activatedperipheralbloodlymphocytes,hRANTESwascloned,sequencedandexpressedinvitro,andmRANTESwasdirectlysequencedforhomologycomparison.Results:Anexpected276bpfragmentwasobtainedinbothamplifications,andsequencedatademonstratedarelativelyhighhomologyamongdifferentcopiesofhRANTES(97%),andhRANTESwasupto95.6%homologoustomRANTES.WhencomparedwithRANTESfromothermammals,hRANTESgaverisetoahomologyrangingfrom77%to86%.TheclonedhRANTESwasexpressedinvitroandapositivesignalofRANTESwasdetectedbydotblotting.Conclusion:Thefull-lengthofhRANTESsequencewassubmittedtoGenBankandhadbeenreleased.OurmRANTESsequenceisfirstreportedandnotyetappearedinGenBank.ThesuccessfulcloningandexpressionofhRANTESwillprovideabasisforAIDSgenetherapyinthefuture.

简介：Objective:TostudytheimmunologicalmechanismsofCondylomaacuminata(CA)throughinvestigatingTlymphocytesubsetlevelsandcytokineprofileintheperipheralbloodofpatientswithCondylomaAcuminata.Methods:Tricolorandbicolorimmunofluorescentstainingantibodyofcellsurfaceantigenandintracel-lularIL-2,IL-4,IL-12,IFN-γinCD4^+andCD8^+T-lymphocytesfrom20patientswithCAwereperformedandfollowedbyflowcytometry.Results:ThenumberofCD3^+T,CD4^+T-lymphocytescellsandCD4^+/CD8^+Tcellsratioweresignificantlydecreased(P<0.01)inpatientswithCAComparedtocontrols,andIL-2,IL-12,IFN-γproductioninCD4^+Tcellswasdecreased(P<0.01),IL-4andIFN-γproduc-tioninCD4^+Tcellswasnotsignificantlydifferent(P>0.05),whileIL-2andIL-12productioninCD8^+Tccellswasdecreased(P<0.01),whereasIFN-γandIL-4pro-ducinginCD4^+Tcellswereofnosignificantlydifference(P>0.05).Conclusions:TherewasanimbalanceofTlympho-cytesubsets,Th1/Th2cytokinesandTc1/Tc2intheperipheralbloodofCApatients,whichmayplayanimportantroleinthepathogenesisandprogressionofCA.

简介：Objective:TostudythebiologicalactivityofMycoplasmapenetrans35kDalipoprotein(P35)invitro,prokaryoticexpressionvectorpQE31/p35wasconstructedandrecombinantfusionproteinP35(rP35)wasexpressedinE.coli.Methods:Thep35genewasamplifiedbypolymerasechainreaction(PCR),clonedtopQE31,andapositiveclonewasscreened.PCR-mediatedmutagenesiswasusedtochangethetwo"TGA"tripletsto"TGG"tripletswithinthep35gene.ProductionoftherecombinantproteinwasinducedbytheadditionofIPTGtotheE.coliculture,rP35waspurifiedwithaNi-NTASpinKitandrP35purificationwasanalyzedbyWesternblot.Results:About1KbPCRamplificationwasclonedintopQE31.Thetwo"TGA"tripletswithinthep35geneweresuccessfullychangedto"TGG"triplets.ThepQE31/p35vectorexpressedaproteinwithacalculatedmolecularmassof37.4kDainE.coli.Westernblotindicatedthe37.4kDaproteinwasrP35.Conclusion:PQE311p35,aprokaryoticexpressionvectorcontainingp35gene,wassuccessfullyconstructedandexpressedinE.coli.

简介：Objective:Toinvestigatetherelationshipbetweenapoptosisandproliferatingcellnuclearantigen(PCNA)expressionofkeratinocytesinCondylomataacuminata(CA).Methods:PCNAexpressionwasobservedbyimmunohistochemistrytechnique(ABCmethod)in51CAspecimensand18normalspecimensofforeskinorvaginalmucosae.55specimens(40intheCAgroupand15inthecontrolgroup)wererandomlysampledforinsitulabelingofapoptoticcellsusingtheTUNELmethod.Results:PositiveexpressionofPCNAinCAandcontrolgroupswere90.2%and77.8%,respectively,andtheproliferationindexinCAgroupwassignificantlyhigherthanthatinthecontrolgroup(P<0.001).Thepositiverateofapoptosiswas42.5%intheCAgroupand53.3%inthecontrolgroup,andtherewerenosignificantdifferencesintheapoptoticindexandapoptosis-proliferationratiobetweentwogroups(P>0.05).Theproliferationindexshowedasignificantnegativecorrelationwiththeapoptosis-proliferationratio(r=-0.62,P=0.01)intheCAgroup.Conclusion:ItissuggestedthattheproliferativeappearanceofCAcouldbeduetotheimbalancebetweencellgrowthandcelldeathwhichiscausedbymoreproliferationandlessapoptosisinkeratinocytes.

简介：Objective:ToevaluatethehumoralimmuneinductioninratsofacandidateAIDSvaccineexpressingthegagp24genefromasubtypeBHIV-1isolate.Methods:Theamplifiedp24genewasinsertedintoaneukaryoticexpressionvectortoformthesupercoiledDNAvaccine.ThelinearizedexpressedDNAvaccinewaspreparedfromtheexpressionplasmidbypolymerasechainreaction(PCR).TheantigengeneexpressioninratsofthelinearizedandsupercoiledDNAvaccineswereinvitroandinvivodetected.Results:InvitrotranscriptionandNorthernhybridizationshowedthatthelinearizedDNAvaccinecouldsynthesizeamountsofp24mRNAsimilartothesupercoiledDNAvaccine.AntibodyassaysofinoculatedratsconfirmedthatthelinearizedexpressionDNAcouldinduceaslightlyhigherantibodytiterthantheexpressionplasmid,whilethehighestantibodytiterhadbeeninducedbyplasmidplusadjuvantinoculation.Conclusion:TheconstructionofacandidateAIDSvaccinebasedonthep24genecouldshedlightonapotentialHIVvaccine,meritingevaluationinarhesusmacaqueSHIV-AIDSmodel.

简介：Objective:Toconstructarecombinantplasmidcontainingtheoutermembraneprotein2(Omp2)geneofChlamydiatrachomatisandexpressOmp2inE.coli.Methods:Theomp2geneofC.trachomatisserovarDwasclonedintopQE30vectorfollowingPCRamplificationfromgenomicDNA.E.coliM15transformantswereinducedtoexpressthefusionproteinbyIPTGandtheproductwasidentifiedbySDS-PAGEandWesternblot.Results:ConfirmedbyenzymecleavageanalysisandDNAsequencing,acorrectrecombinantplasmidpQE30/omp2wasconstructed.Thefusionproteinfromthetransformantswasapproximately60kDainsizeinSDS-PAGEanalysis,whichcouldspeciallyreactwithanti-6×HismousemonoclonalIgGantibodies.Conclusion:WesuccessfullyexpressedOmp2inE.coilM15,providinganefficientandsimplesystemforassayingtheimmunologicalpropertiesofOmp2.

简介：Objective:ToobtainrecombinantTreponemapallidumsubsp,pallidum(TP17KD)lipoproteininlargequantitiesbyamplificationandtofurtherpurifyantigensforlaboratorydiagnosisofsyphilisanddevelopmentofasyphilisvaccine.Method:TheTppl7lipoproteingenewasamplifiedfromtheTP(strainNichols),andthenitwasrecombinatedintoaplasmidpMAL-2candclonedwithinE.coli12-TB1.ThehostbacteriacontainingrecombinantplasmidswereinducedwithIPTG.TheTpp17KDlipoproteingenewasamplifiedbyus-ingPCRandpositivecloneswerescreenedwithdoubledigestionandPCR.RecombinantplasmidsweretransformedintoE.coliandtheE.colicarryingrecombinantplasmidswereinduced.TheexpressionofTP17KDwasdetectedbysodiumdedecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)andimmunoblot.Results:GelstainingwithCoomassieblueG-250showedthattheinducedE.colicarryingrecombinantplasmidcouldproduce60KDfusionproteinathighlevels.Gelscanningshowedthat17KDproteinexpressioninE.coliaccountedfor10%oftotalcellularprotein.Therecombinantproteinantigenreactedwiththeseraofsyphilispatients.Conclusion:Ourstudylaysacornerstonefordevelopingnewtechniquesoflaboratorydiagnosisforsyphilisandnewvaccines.Preliminaryclinicalapplicationshowedthatthefusionproteincouldbeusedforthediagnosisofsyphilis.

简介：Objective:Todetecttheactivatedexpressionoftelomeraseincondylomaacuminatalesionsinlow-risk(6/11)andhigh-risk(16/18)humanpapillomavirus(HPV)infectionandexaminetheroleplayedbytelomeraseintheoccurrence,developmentandcarcinomatouschangeofcondylomaacuminata.Methods:AssayingtheexpressionoftelomeraseandthetypeofHPVindamagedskinof42CApatientsandnormalskinof30healthycontrolindividualsthroughtelomeraserepeatamplificationprotocol(TRAP)andpolymerasechainreaction(PCR).Results:Inallthenormalskincontrols,PCRforHPVwasnegativeandonly16.7%ofsampleswerepositiveforTLMAexpression;inCAlesions,HPVtestingwaspositiveinall(32caseswerelow-risk,3werehigh-risk,and7wereofmixedtype)andallwerepositiveforTLMAexpression.Conclusion:TLMAmaybeactivatedbyHPVinfection,andinturncausethehyperplasiaofepidermalcells.ItwasalsoindicatedthatHPV,especiallyhighrisktypes,canactivateTLMA.TheactivationofTLMAmayplayanimportantroleinabnormalhyperplasiaandcarcinomatouschangesinCAlesions.

简介：Objective:Toamplifyantigengenesfrompatientswithhumanimmunodeficiencyvirustype1(HIV-1)inGuangdongProvinceforcandidateAIDSvaccinedesign.Methods:Viralnucleicacidwasisolatedfrom10HIV-1infectedindividuals'peripheralbloodcollectedduring1995-2000inGuangdongProvince.Theviralgagp24geneandenvgp120genewereamplifiedbynested-PCRandsequenced.ThehomologiesamongHIV-1isolateswerecomparedwithHIV-BLAST.Results:Among10HIV-1isolates,ninearehomologoustovirusesofsubtypeB,andoneishomologoustovirusesofsubtypeE.Conclusion:SubtypeBvirusesofHIV-1arepredominantlypresentinGuangdongProvince.

简介：Objective:TostudytheexpressionofFasandBcl-2proteinsonTlymphocytesubsetsintheperipheralbloodofrelapsingpatientswithcondylomaacuminatum(CA)andhealthycontrols.Methods:Flowcytometry(permeabizationandstainingprocedurewithconjugatedantibodies)wasused.Results:WeobservedthattheexpressionofFasproteinonCD4^+TlymphocytesubsetofCApatientswassignificantlyhigherthanthatofhealthycontrols(P<0.01).Conclusions:IncreasedexpressionofFasproteinonCD4^+Tlymphocytesubsetmaybeacauseofde-creasedpercentageofCD4^+Tlymphocytesubset.ThisinducestheincreasedratioofCD4^+/CD8^+.

简介：Objective:Tostudytheexpressionofactivatedepi-dermalgrowthfactorreceptor(EGFR)andtranscrip-tionfactorE2F(E2F)inCondylomaAccuminata(CA)patients.Methods:ImmunofluorescenttechniqueswereusedtoinvestigatetheexpressionofactivatedEGFRandE2FinCApatients.Results:TheexpressionofactivatedEGFRonthemembraneofepithelialcellsinCAlesionswassig-nificantlygreatercomparedtoexpressionleversinthecontrolgroup(P<0.01).Moreover,theco-expres-sionofactivatedEGFRandE2Fwassignificantlyin-creasedcomparedtothecontrolgroup(P<0.01).Conclusion:Ourobservationssuggestthatthein-creaseinactivatedEGFRexpressionmaystimulatehyperplasiainCApatientsthroughtheactivationoftranscriptionfactorE2F.

简介：

简介：Abstract:Thisstudyinvestigatedtherelationshipbetweenhumanpapillomavirus(HPV)genotypeandexpressionofp53andp21^WAH1.Expressionofp53andp21^WAH1in35casesofcondylomaacuminatumspecimensinfectedbyHPV6/llandHPV16/18werestudiedusingimmunohistochemicalstaining.Allspecimensofthecondylomaacuminatumcasewerepositiveforexpressionofp53andp21^WAH1.Theexpressionofp53incondylomaacuminatuminfectedbyHPV16/18wassignificantlylowerthanthatinspecimensinfectedbyHPV6/ll.However,expressionofp21wAHbetweenthetwogroupswasnotsignificantlydifferent.Expressionofp53incondylomaacuminatumislikelyrelatedtoHPVgenotype,expressionofp21^WAH1wasnotrelatedtoHPVgenotype.

简介：Objective:Tostudytheroleofmonocytesinthepathogenesisofgenitalherpes.Methods:TNF-aandIL-6levelsin27casesofgenitalherpesweredetectedbyenzymelinkedimmunosorbantassay(ELISA).HLAclassⅡantigenexpressiononmonocytesweredetectedbyanalkalinephosphataseanti-alkalinephosphatasemethod.Results:Comparedwithnormalcontrols,levelsofTNF-aandIL-6secretedbymonocytesrespondingtoLPSmitogeninvitroweresignificantlydecreased[(3.13±0.44ng/ml)vs(4.68±0.54ng/ml),P<0.05and(3.32±1.06ng/ml)vs(6.46±1.94ng/ml),P<0.05,respectively].HLAclassⅡantigenexpressiononmonocytesinthegenitalherpesgroupwasalsosignificantlydecreased[HLA-DR(67.48%±1.51%)vs(81.03%±1.32%),P<0.01andHLA-DQ(29.54%±1.15%)vs(37.63%±1.79%),P<0.01respectively].Conclusion:Thesefindingssuggestthatthedecreasedmonocytefunctionmaycontributetothepathogenesisofgenitalherpes.Augmentingorinducingmonocytefunctionmaybeimportantintheprevention,treatment,andreductionofgenitalherpescases.

简介：Objective:ToinvestigatetheexpressionofHPV16mRNAinnormalhumankeratinocytestransfectedwithpSV2-neo/16.Firsthumankeratinocyteswereculturedintheserum-freemediumM154.Second,theplasmidpSV2-neo/16wastransfectedintothehumankeratinocytesusingatransfectingreagent.Third,RT-PCRandSouthernBlottingwereusedtodetecttheexpressionofHPV16mRNAandDNAinthetransfectedkeratinocytes,respectively.Results:TheexpressionofHPV16mRNAwassuccessfullyamplifiedandan110bpwasditectedbyRT-PCR.A7.9kbfragmentwasconfirmedinthetransfectedkeratinocytesbySouthernBlotanalysis.Conclusion:HPV16mRNAandDNAweresuccessfullydetectedinthehumankeratinocytes.

简介：Objective:Toanalyzesubtypesandquasi-speciesofisolatedvirusesfromHIV-1infectedindividualsamongthepopulationepidemiologicaldynamicsoflocalHIV-1isolates,thuslayingafoundationfordesigningacandidateAIDSvaccine.Methods:Byhetero-duplexmobilityassay(HMA)andsinglestrandconformationpoly-morphism(SSCP)analysisonampliconsfromsingle-primedpolymerasechainreaction(SP-PCR),subtypesandquasi-speciesoftestedHIV-1isolateswereelucidated,andampliconsweresequencedforconfirmation.Results:Specificampliconsfromdifferentsubtypesandquasi-speciesofHIV-1couldbediscerniblebyHMAandSSCPanalysis.HIV-1isolatesfromdifferentpatientsmightbeeitheradifferentsrbtypeoranidenticalsubtype,andHIV-1isolatesfromanindividualwerepresentinapopulationofquasi-species.