简介：Chlamydiatrachomatisoutermembraneprotein2(Ctomp2)isamajorimmunogeninchlamydialinfectionsandahighlygenus-conservedstructuralproteinofallChlamydiaspecies.Topurifytheproteinandtopreparemonoclonalantibodies(mAbs)againstit,therecombinantproteinwasinducedbyIPTG,whichwasconfirmedbySDS-PAGEandpurifiedbymeansofaNi2+-chargedresincolumn.ThedenaturedproteinwasrefoldedintheGSH-GSSHbuffergraduallyandidentifiedbyWesternblotting.ThentheBALB/cmicewereimmunizedwiththerecombinantproteintopreparethemAbagainstCtomp2.TheobtainedmAbswerecharacterized.GenitalspecimensweretestedwithindirectELISAmostlymadeofthemAbandcellculturein84patientswithgenitalsymptoms.Theresultsshowedthathigh-levelexpressionoftherecombinantproteinwasachieved,whichexistedasinclusionbodyandamountedto38%oftotalbacteriumprotein.AmAbagainstCtomp2wasobtained.ItbelongstoIgG2b.Thetiterswereashighas1:40000.TheWesternblottingshowedthatthemAbonlyreactedwiththerecombinantprotein.IthadnocrossingreactionsagainstE.coli,N.gonorhoea,M.hominis,U.urealyticumandM.penetrans.Ithadhighspecifity.Incomparisonwithgoldstandardtest-cellculture,thesensitivities,specificities,positivepredictivevaluesandnegativepredictivevaluesofindirectELISAwere95.24%,100%,100%and98.44%,respectively.Theabove-mentionedresearchworkcontributednotonlytothefurtherstudyofthestructureandfunctionofthisprotein,butalsototheestablishmentofthemethodforitsclinicalapplication,forithadnotbeenreportedbefore.

简介：AThepurposeofthisinvestigationwastostudythetherapeuticeffectofLamivudineonHBVDNAinperipheralbloodmononuclearcells(PBMC)andserum,andthelevelofcytokinesinserumofthepatientswithchronichepatitisB.Thepatientsweredividedintotwogroups(A=47,B=34),andtreatedbyLamivudine,routinemedicine,respectively.ThelevelsofHBV-DNAinPBMCandserumandcytokineswerealldetectedbeforeandaftertreatment.AfterthetreatmentofLamivndinefor36weeks,thetotalconversionnegativeratesofHBV-DNAinPBMCandserumofthepatientstreatedwithLamivudinewere55.32%(26/47)and61.70%(29/47),respectively.ThetotalnegativeconversionratesofHBV-DNAinPBMCandserumofthepatientstreatedbyroutinemedicinewere26.47%(9/34)and32.35%(11/34),respectively.TherewassignificantdifferencebetweenLamivudinegroupandroutinemedicinegroup(P<0.01).ThenegativeconversionratesofHBeAginserumofthepatientswere46.81%(22/47)and68.09%(32/47)attheendof24weeksand36weeks,andwerehigherthanthoseofroutinemedicinegroup(P<0.05andP<0.01).Thelevelsofalanineaminotransferase(ALT),aspartateaminotransferase(AST),ALT/ASTinserumofthepatientsafterbeingtreatedbyLamivudine,routinemedicineweredown-regulatedto(30.1±9.6)U/ml,(32.3±10.7)U/ml,0.9±0.1and(48.4±10.7)U/ml,(44.7±11.0)U/ml,1.1±0.2.Aftertheanalysisofvariance,thehighsignificantdifferencewasobviousbetweenthetwogroups(P<0.01).ItwasduetothehighlevelsofIL-6,IL-8andTNF-αinchronichepatitisBwhichcouldbedown-regulatedto(250.5±33.3)pg/ml,(153.4±22.2)pg/ml,(232.6±21.2)pg/mlbyLamivudine,whichwasmoreobviousthanthatofroutinemedicine(P<0.01).LamivudinehashightherapeuticeffectonthetreatmentofHBVDNAinPBMCandserumandhasbettertherapeuticeffectthanthatofroutinetherapy.Lamivudinemayalsohavehigherdown-regulatedinflammatoryinfiltrationandsecretioninlocalsitecaused

简介：TheaimofthisstudywastoexplorethemolecularbasisfortheattenuationoftheJapaneseencephalitisvirus(JEV)vaccinestrainSA14-14-2.ThevirulenceofSA14wildJapaneseencephalitisvirus(JEV)anditsseveralattenuatedviruseswastestedbyintracerebral(i.c.)orintraperitonial(i.p.)inoculationof10-12gmice.Thestabilityofneuroattenuationwastestedbyonepassageinsucklingmousebrain.TheEproteingenesofthoseviruseswereamplifiedbyPCR,sequencedandcompared.Threeattenuatedvirusstrains,SA14-14-2vaccinevirus,SA14-9-7andSA14-5-3,didnotexhibitlethalinfectionsbyi.c.ori.p.inoculationof10-12gmiceandreverttothevirulence.Theothervirusstrain,SA14-12-1-7,showednoneuroinvasivenessbyi.p.inoculationbutresidualneurovirulencebyi.c.inoculationandrevertedtohighvirulenceafteronebrainpassage.ComparisonoftheEproteingenesequencesofthefivevirusstrainsindicatedthatthereweredifferencesoftwelvenucleotidesandeightaminoacidsbetweentheparentstrainSA14andvaccinestrainSA14-14-2,ofwhichsixaminoacids(E-107,E-176,E-439,E-138,E-279,E-315)exhibitedchangescommontothoseofSA14-9-7andSA14-5-3,threesubstitutionscommontoSA14-12-1-7.TwoaminoacidsubstitutionsatthesitesE177(T→A)andE264(Q→H)areuniquetotheSA14-14-2vaccinevirus.TheresultssuggestthatthemutationsofE-107(Leu→Phe),E-176(He→Val),andE-439(Lys→Arg)maycontributefortheattenuationofneuroinvasivenessandpartiallyfortheattenuationofneurovirulence,themutationsofE-138,E-279,E-315maynotonlycriticaltotheneuroattenuationbutalsotoitsstability.

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简介：TocloneandexpresstherecombinantoutermembraneproteinTp0453ofTreponemapallidumandtoanalyzetheimmuno-reactivityandimmunogenicityoftheexpressedprotein,theimmuno-dominantepitopeoftheTp0453wasamplifiedfromthecompletegenomeofT.pallidumbyPCR,subclonedintoexpressionvectorpQE32togeneratetherecombinantplasmidpQE32/Tp0453,thenexpressedinE.coliM15andanalyzedbySDS/PAGEandWesternblotting.ThefusionproteinexpressedwaspurifiedwithNi-NTAaffinitychromatography.Itsimmuno-reactivitywasassayedbyindirectELISA,andtheimmunogenicitywasdeterminedbyimmunizationwiththisfusionproteininNewZealandrabbits.Inthepresentstudy,afusionproteinofmolecularweightabout32kDawasobtained.AsdemonstratedbyWesternblotting,therecombinantproteincouldreactspecificallywithpositiveIgGseraofpatientswithsyphilis,andtheantibodiesagainstT.palliduminhumanseraweresuccessfullydetectedbyindirectELISA.BoththesensitivityandspecificityofELISAbasedontheTp0453fusionproteinaswere100%(30/30)whendetectedwithcontrolsera.IncomparisonwiththeresultsofIgGELISAwiththoseofTPPA.ItwasfoundthatthesensitivityofELISAwas96.8%andthespecificitywas100%.ThedifferenceofELISAandTPPAwasnotsignificant,andtheconcordanceofresultsbetweenELISAandTPPAwas98.2%.Inaddition,specifichumoralresponsescouldbeelicitedbyimmunizationwiththerecombinantfusionproteininNewZealandrabbitswithaspecificantibodytiterof1:1280after3successivedosesofimmunization.Theseresultsdemonstratethattheexpressedrecombinantfusionproteinshowsexcellentimmuno-competenceandprovidefoundationtodevelopaquickdiagnostickidappliedtodetectthepresenceofT.palliduminfections.

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简介：Thepurposeofthestudyistoestablishafluorescencequantitativereversetranscriptionpoly-merasechainresponse(FQ-RT-PCR)methodforthequantitativedeterminationofIL-2mRNAandIL-4mRNAinThcells,withwhichtheThcellsstatusofthepatientswithgynaecologicaltumorsandchronicrenalfailure(CRF)canbeanalyzed.IL-2cDNAandIL-4cDNAwereprepared,andtheplasmidpMD18carryingIL-2cDNAorIL-4cDNAfragmentwasconstructedandclonedasthetemplateforquantitativedetermination.Theprimersandprobeslabelledwith6-carboxy-fluorescein(FAM)and6-carboxy-tetrarnethylrhodamine(TAMRA)wereprepared,andtheexperimentalconditionswereoptimizedtosetuptheFQ-RT-PCRmethodforquantitativedeterminationofIL-2mRNAandEL-4mRNA.Thcellsenrichedfromperipheralbloodmononuclearcells(PBMCs)of20healthyvolunteers(HVs),16gynaecologicalbenign(GB)cases,18gynaecologicalmalignant(GM)tumorcasesand16chronicrenalfailure(CRF)patientsweretestedforIL-2mRNAandIL-4mRNAbyFQ-RT-PCR.Thehouse-keepinggeneβ-actinwasusedastheinternalcontrolgeneoftheexperiment.Thestandardcurveforlogconcentrationofseriesofquantitativetemplatesvsthresholdcycle(CT)wasestablishedbylinearregression,andthelinearrangewas102-107copies/μl.TheimprecisiontestshowedtheCVofinter-assayandintra-assayofahighcontentsamplebyFQ-RT-PCRwere7.8%and12.5%,respectively.TheCVofinter-assayandintra-assayofalowcontentsamplewere10.8%and19.5%,respectively.TheIL-2mRNAexpressionsinThofthepatientswithgynaecologicalmalignanttumor(comparedwiththeHVsandthepatientswithgynaecologicalbenigndisease)andinThoftheCRFpatients(comparedwiththeHVs)weredeclinedsignificantlyandatthesametimetheIL-4mRNAexpressionincreasedsignificantly(P<0.001).Asimple,sensitiveandaccurateFQ-RT-PCRmethodforthequantitativedetectionofIL-2mRNAandIL-4mRNAhasbeenestablished.TheIL-2mRNAandIL-4m

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：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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