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简介：Inordertoanalyzethesequencesoftheinternaltranscribedspacer(ITS)includingthe5.8SribosomalDNA(rDNA)ofcommondermatophytes,soastoobtainarapidandaccuratemethodtoidentifythespeciesofdermatophytesandtoestablishthephylogenetictreeofthesespeciestounderstandtheirrelationship,16strainsofdermatophyteswerecollectedandpreliminarilyidentifiedbymorphologicalcharacteristics.GeneralprimersforfungiITS1andITS4wereusedtoamplifytheITSrDNAofeachstrainswithPCR.ThePCRproductsafterpurificationweresequenceddirectlyandwereanalyzedthroughinternet.Intheresults,11strainswereidentifiedbymeansofmorphologicalfeatures,amongwhich5strainswereTrichophyton,5strainswereMicrosporumand1wasEpidermaphyton,whichwasconsistentwiththeresultsbymolecularbiology.Inthe5unidentifiablestrains,1strainwasprovedtobeChrysosporiumbymolecularbiology.Thesestrainsstudiedcouldbedividedinto3differentclassesasindicatedintheanalysisofthephylogenetictreeofthesequencesinITS,whichwerequitedifferentfromthoseofmorphologicalclassification.ItisevidentfromtheaboveobservationsthatthemolecularmethodofanalysisontheITSsequencesisarapid,highlysensitiveandaccurateapproachforthedetectionofdematophytespecies,however,itstillexhibitssomelimitationsneedingthesupplementationwithmorphologicalidentification.

简介：Tosupportthescientificbasisforrapididentificationofpathogenicbacteriaandotherstud-ies,thesequencesofhsp60geneinmajor34speciesof16genusofpathogenicbacteriaweresearchoutinGenBankandaproperpairofuniversaldegenerateprimerwasdesignedbymeansofthemolecu-larbiologicalsoftwawePrimer5.0andOligo6.0.ThisprimerwasthenusedinthePCRamplification,andthehsp60genefragmentsoftheselectedpathogenicbacteriacouldbeamplifiedusingthisdegener-ateprimer.Bywayofbioinformationalanalysis,theconservation,variationandtheinterspeciesphylo-geneticrelationsofthehsp60genesequencewereanalysed.Fromtheresultsofthecomparativestudyonsequences,itwasdemonstratedthatthehsp60genewascharacterizedbyconservationandvaria-tion,inwhichtheconservedandmutantregionsco-existedandseparatelydistributedwithmanysmallmutantregionsdistributedamongtheconservedregions,justlikethemosaic.Thephylogenetictreeamongdifferentpathogenicbacteriadrawnfromthehsp60geneanalysiswasprovedtobeconsistentwiththosefrom16SrRNAand23SrRNA.Itisconcludedthatthesequencedistributionofhsp60genewouldprovideasolidbasisfortherapididentificationofpathogenicbacteriaandthedevelopmentofadiagnosticmicroarray.

简介：TheaimofthisstudyistoexplorethegenomicmolecularorganizationandgenogroupofhumannorovirusfrominfectedinfantsinGuangzhouofChina.PrimersweredesignedaccordingtothegenomicsequenceofnorovirusintheGenBank,andthenorovirusgenomewasamplifiedbyRT-PCR.ThePCR-productswereclonedintoTvectorandsequenced,andthegenomicnucleotidesequenceswereanalyzedwiththeprogramsCLUSTALW/X,DNASTARandRAT(RecombinationAnalysisTool).TheNVgz01straingenomeis7558bpinlengthandencodesthreeopenreadingframes(GenBankaccessionNo.isDQ369797).ThegenomicsequencesofNVgz01werecomparedwiththoseofnomvirusinGenBank,whichrevealedthatthehomologywithgenogroupⅡrangesbetween76%-90%,andgenogroupⅠbe-tween43%-44%.TheORF1regionshared94%and88%identitywithMc37andFarmingtonstrains,respectively;thecapsidregion(ORF2)shared65%and94%identitywithMc37andFarmingtonstrains,respectively.Phylogenetictreeswerereconstructedbytheneighbor-joiningmethod.ComparativecompletesequenceanalysisoftheNVgz01withreportedhumannorovimsgenomicsequencesrevealedthatthisisolatebelongstogenogmupⅡ.TheORF1andORF2regionsshareddifferentidentitywithMc37andFarmingtonstrains,suggestingNVgz01couldbearecombinantvires.

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简介：ToanalyzethegenomicmolecularstructureandgenotypeofhumanastrovirusisolatedfrominfantinGuangzhouofChina,theprimersweredesignedbasedonthegenomicsequenceofastrovirusfromtheGenBankandthetargetsequencewereamplifiedbyRT-PCR.ThenthePCR-productswereclonedtoTvectorandsequenced.ThegenomicnucleotidesequenceswereanalyzedbytheprogramsCLUSTALWandDNASTAR.ItwasfoundthatthefullgenomiclengthofHASTVgz01strainwas6721bpandtheORFswere6558bp.The5'and3'UTRwere82and81nucleotides.Thegenomeincluded3openreadingframes(ORFs):ORF1a,ORF1bandORF2.The5'-terminalORF1astartedatnucleotide83andextendedtonucleotide2845.ORFlb(nt2785tont4332)overlapedORFlaby61nucleotides.The3'-terminalORF2beganatnucleotide4325andterminatedatnucleotide6640.ORF2had2316nucleotides.ComparedwithotherastrovirussequencesinGenBank,thehomologyoftheaminoacidsequenceofORF2ofHASTVgz01strainwiththatofserotype4was93%.Homologywithotherserotypesrangedfrom61%to70%.ThecompletenucleotidesequenceofastrovirusHASTVgz01strainisolatedfromGuangzhouinChinawas6721bpinlength,GenBankaccessionNO.DQ344027.ComparingtheORF2ofastrovirusHASTVgz01withtheknownsequencesoftypes1-8thehighesthomologywasserotype4(93%).ComparativesequenceanalysisoftheHASTVgz01ORF2withthereportedhumanastrovirussequencesrevealedthattheisolatedastrovirusbelongstogenotype(serotype)4.

简介：Thepurposeofthisinvestigationistostudytheclinicalcharacteristicsofinfectionsbycommunity-acquiredmethicillin-resistantStaphylococcusaureus(MRSA)andtheconditionofantibioticsresistanceoftheclinicalisolatesinordertoguidefortherationaluseofantibiotics.Withtheclinicalisolatesfromcasesofhospital-acquiredMRSAatthesameperiodascontro|s,theclinicalcharacteristicsofinfectionsbycommunity-acquiredMRSAinHangzhouareaandthepatternofnon-β-lactamaseantibioticsresistanceweredeterminedinthisstudy.Itwasfoundthattheaverageageofpatientswithcommunity-acquiredMRSAinfectionswas30.89+13.3,incomparisonwiththoseofthehospital-acquiredpatientsof56.0+11.8,appearingtobeyoungerthanthoseofthelatter,andtheformershowingnoanybasicillness.Bothoftheformerandthelatterweresensitivetovancomycin(100%vs100%),andtheyhadthesamedegreesofsensitivitytorifampicin,fosfomycin,andSTM/TMP(86.8%vs88.1%,P>0.05;81.6%vs82.9%,P>0.05;and52.6%vs61.9%,P>0.05,respectively).Theformerwasmoresensitivetonetimycin,chndamycin,erythromycinandminocyclinethanthoseofthelatter(73.7%vs50.5%,P<0.01;60.5%vs45.7%,P<0.05;28.9%vs11.4%,P<0.01;and81.6%vs58.6%,P<0.01respectively).Meanwhile,theincidenceofmulti-resistantstrainofisolatesintheformerwassignificantlylowerthanthatofthelatter(31.6%vs81.0%,P<0.01).Inconclusion,itappearsthatthestrainsofclinicalisolatesisolatedfrompatientswiththecommunity-acquiredMRSAinfectionsshowdifferentclinicalcharacteristicsandantimicrobialsusceptibilityincomparisonwiththoseofthehospital-acquiredcasesofinfection,andthisnecessitatesanalterationinthechemotherapyofinfectionssuspectedtobecausedbycommunity-acquiredMRSA.

简介：InordertoimprovethefunctionalaffinityofthehumanizedVHsingledomainantibodyagainsthumanlungcancer,thegenescodingthehomogenousdimersdihu3D3V_Handtetramerstehu3D3V_HwereconstructedbyfusingtheSVS-Cysshortpeptideandp53tetramerizationstructuraldomaingenetohu3D3V_HgeneviarecombinantPCRtechnique,respectively.Then,thedihu3D3V_Handtehu3D3V_HgeneswereclonedtotheprokaryoticexpressionvectorpET-22b(+)andexpressedinE.coliBL21(DE3).TheproteinsexpressedwerepurifiedthroughNi~(2+)-affinitychromatographiccolumn.Meanwhile,thehu3D3V_H,dihu3D3V_Handtehu3D3V_HproteinswerelabeledwithFITC,andtheirreactivitywithan-tigenandspecificitywereanalyzedbyimmunofluorescenceassay.Astotheirfunctionalaffinities,itwasanalyzedandcomparedbyflowcytometry.Theresultsindicatedthatthesetwogeneswereexpressedasmonomersandmainlyasinclusionbodies.Afterpurificationandrenaturation,therewereabout50%ofdimersand70%oftetramerremainingintheproteinsolution.Inaddition,thedihu3D3V_Handte-hu3D3V_Hproteinsstillremainedthereactivitywithantigenandspecificityofhu3D3V_Hprotein,andtheirfunctionalaffinitieswereincreasedabout60%or100%respectively,comparedwiththoseofhu3D3V_Hprotein.Itisevidentthatthefunctionalaffinityofhu3D3V_Hproteincanbegreatlyimprovedbyincreasingitsbindingvalency.

简介：Inthepresentstudy,thedrug-resistancegenesencodingβ-lactamases,aminoglycosidemodifyingenzymes,DNAtopoisomerasesandintegronaswellastheirmolecularepidemiologywereinvestigatedbymeansofanalyzingthedrug-resistanceandmolecularepidemiologyofAcinebacterbaumanniiisolatedfromtheclinicalsamplesintwohospitalsinQiangzhouandHuzhoucityofJiangsuandZhejiangprovincefromJuly2000toMarch2005.Theminimalinhibitoryconcentrations(MICs)ofthese307isolatesweredetectedbyautomaticmicrobiologicalsystem,and35strainsagainst5-fluoro-quinoloneswereperformedbyagardilutionassay.Meanwhile,theresistantgenesin80isolateswereamplifiedbyPCRwithidentificationbyDNAsequencer.Itwasfoundthatmostofthe307isolatesofA.baumanniiwereresistanttomultipleantibioticstested,inwhichtheresistanceratesoftheisolatesagainstpiperacillin,piperacillin/tazobactam,amoxacillin/clavulanicacid,cefotaxime,ceftazidime,cefepime,gentamicin,amikacin,ciprofloxacin,chloramphenicolandsulfamethoxazole/trimethoprimwereallabove35%,butthoseofimipenemandmeropenemwerequitelow,rangedonly2.6%and3.3%.Inaddition,itwasalsodemonstratedthatthepositiveratesofTEMandSHVβ-lactamasegenesaccountedfor93.8%and22.5%respectively,andthoseoftheaminoglycoside-modifyingenzymegenesincludingaacC1,aacC2,aacC3,aacC4,aacC4A,aphA6,ant(2')-Iandant(3')Iwere58.8%,8.8%,7.5%,28.8%,45.0%,2.5%,28.8%and65.0%respectively.Themutationsinthequinolone-resistantdeterminingregion(QRDR)ofgyrAandparCgenesindicatedthatsubstitutioninSer-83residueofGyrAproteinwasmostfrequentlyoccurredamongstrainswithMICforciprofloxacinofmorethan4μg/ml,whereasadoublemutationatSer-83residueofgyrAandSer-80ofparCwasfoundinstrainswithMICofciprofloxacinofmorethan8μg/ml.Astothepositiveratesofclass1integron(IntI-1)andqacE△1-sul-1,itwasfoundtobe60.0%and77.5%respectively

简介：ToconstructanexpressionvectorcontainingtheE1glycoproteingeneofrubellavirusforthestudyontheeffectofmutationoftheE1geneglycoproteinandtheanalysisofphylogeneticdifferencesofsequences,thegeneencodingtheE1envelopeglycoproteinwasamplifiedfromrubellavirus,JinanstrainJR23,byRT-PCRandligatedintoPMD-18Tvector.TheclonesthatcarriedtheE1genewereidentifiedafteramprselectionandanalysisofrestrictionenzymedigestion.AftersequencingthisgenewasanalyzedbyDanstarandWinstarprograms,andthemapofphylogenetictreewasdrawn.ThecloneofE1glycoproteinwasthusconstructed.ItwasfoundthatthesequencedifferencesbetweenJR23strainandtheTCRBstrainfromJapanandthosebetweenJR23strainandThomasstrainofEnglandwererathersmallwithdifferencevaluesof0.9%and1.2%respectively.YetthosebetweenJR23strainandBRD2strainfromBeijingandthosebetweenJR23strainandXG379strainfromHongKongwerecomparativelylargerwithdifferencevaluesof7.6%and7.3%respectively.ThesequenceofJR23strainwithotherstrainswaslessthan3%excepttheNCstrain(3.7%).ItconcludesthattheconstructionofE1glycoproteingeneoffersanapproachtostudytherelationshipbetweenstructuresandfunctionsofE1geneanditsgeneproducts.Inthephylogenetictree,itshowsthattherearesignificantdifferencesinthesequencesofrubellavirusisolatedinChina,andthismightbehelpfultodevelopaneffectivesubunitvaccine.

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简介：ToinvestigatetheHLA-A,-BallelepolymorphisminHanpopulationofShandongprovinceandtoexplorethepossibilitytofindouttheHLA-A,-B-matchedcordblooddonorsforstemcelltransplantationtobeusedinotherareainChina,5844umbilicalcordbloodsamplesweretakenfromHanpopulationdonorsofShandongprovince,andassayedwithPCR-sequence-oligonucleotide(PCR-SSO)assay.InShandongHandonors,20allelesatHLA-Alocusand46allelesatHLA-Blocuscouldbedetectedasrevealedinthepresentstudy.Amongthe20allelesatHLA-Alocus,themostprevalentfiveallelesincludedA*02(0.3041),A*11(0.1443),A*24(0.1434),A*30(0.0975)andA*33(0.0859),while,thealleleswithlowergenefrequenciesincludedA*34(0.0006),A*25(0.0005),A*66(0.0005),A*74(0.0004)andA*(0.0001).Ofthe46HLA-Ballelesdetected,themostprevalentfivealleleswereB*13(0.1348),B*51(0.0713),B*62(0.0712),B*61(0.0676)andB*60(0.0642);whilealleleswithlowergenefrequenciesincludedB*77(0.0001),B*76(0.0002),B*47(0.0003),B*42(0.0003)andB*72(0.0004).IncomparisonwiththoseoftheotherHanpopulationinChina,theHLA-A,-BgenefrequenciesintheumbilicalcordbloodofShandongprovincepossessuniquedistributionfeaturesamongtheinvestigatedpopulationsfromvariousregionsofthesameraceorigin,andthedifferencesinvariousregionsofthesameracewerelessthanthoseamongthedifferentrace.ItisevidentthattheHLA-A,-BallelesoftheumbilicalcordbloodtakeninShangdongprovinceshowhighdegreeofpolymorphism,anditmightbepartofthoseofNorthernHanpopulationinChina.So,itisreasonableforpatientsofNorthernChinesetoreceiveHLAclassI-matchtransplantofcordbloodstemcellsfortissueandorgantransplantationfromShangdongumbilicalcordbloodbank.

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.

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简介：Toclonethegenecodingtheimmunodominantregioninthechlamydialprotease-likeactivityfactor(CPAF)fromChlamydophilapneumoniae,toanalyzeimmunocompetenceoftheexpressedprotein,andtoevaluateitsvalueinserodiagnosis,theCPAFimmunodominantregiongenewasamplified,ligatedintoapGEX6p-2vector,andthentheexpressedrecombinantproteinwaspurifiedwithglutathioneS-transferase(GST)agarosegelFFafterrenaturation,thenidentifiedbySDS-PAGEandWesternblot.AnewindirectELISAwasdevelopedwiththepurifiedproteinascoatingantigen.TheimmunogenicityoftherecombinantproteinwasevaluatedbyimmunizationtoNewZealandrabbits,anditsimmunoreactivitywasanalyzedbyreactingwithanti-C,pneumoniaeantibody.300clinicalserasampleswererespectivelyde-tectedbymicroimmunofluorescence(MIF)asreferencemethodandtheindirectELISA,andthediffer-encebetweenthetwomethodswasanalyzed.Cross-reactivityagainstChlamydiatrachomatiswasinvesti-gatedwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Theresultsindicatedthata51.3kDarecombinantproteinwasobtained.Westernblotassayprovedthattherecombinantproteincouldmerelyspecificallyreactwithhumananti-C.pneumoniaeantisera.ThetitersofthespecificIgGan-tibodiesintheimmunizedNewZealandrabbitswereabove1:16000.Anti-C.pneumoniaeIgGpositiveandnegativereferencesereweredetectedwiththeindirectELISA,andtheconcordancerateofnegativeandpositiveresultswereboth100%(40/40).ThesensitivityandspecificityoftheindirectELISAincomparisonwithMIFwere93.8%(45/48)and100%(252/252)separatelybydetecting300clinicalserasamples,andtheconcordanceratebetweenthetwomethodswas99.0%.NocrossreactionagainstC.trachomatiswasfoundwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Incon-clusion,thepreparedrecombinantproteinoftheCPAFimmunodominantregionshowsexcellentimmuno-competenceandcanbeusedtodevelopanewindirect

简介：TherRNAgeneticlocusisfoundinallprokaryoticorganisms,andishighlyconservative,althoughitsrelativelystablevariationsarefoundfrequentlyindifferentbacteria.Theutilityofthislocusasataxonomicandphylogenetictoolhasbeenreportedwidely.Thisstudy,aimedat16SrRNAgene(16SrDNA)andwiththehelpofbiomolecularmethods,attemptedtoachievethegoalofrapididentificationofcommonpathogensInthisstudy,333clinicalisolatedpathogenicbacteriawerecollected。TwopairsofprimerswerechosenandlabeledwithdifferentfluorescentdyesandthenusedtoamplifythegenomicDNAextractedfrombacteria.ThePCRproductswerethendetectedbycapillaryelectrophoresis-singlestrandconformationpolymorphism(CE-SSCP).Inordertopursuehigherresolutionandpeak-separationeffect,ahighefficientseparatingmedium,linerpolyacrylamidedel(LPA),wasputtouseinthisstudy.Finally,everybacteriacolonygenerateddistinctpatternsfromeachother,whichwereeasilytobeusedforidentification.TheseresultsindicatedthatPCR-CE-SSCPwasarapididentificationmethodforbacterialidentification,withtheaspectsofhighefficiencyandhighprecision.Comparedwithtraditionalmethod,thistechnologyisofgreatutilityforclinicaluseespeciallyforitshighsensitivity.