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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.

简介：ThegenomeoftheenterohemorrhagicEscherichiacoliO157:H7EDL933contains177“O”-islands(OIs).TostudytheirpotentialcontributiontotheO157-specificpathogenicity,wesurveyedthedistributionof22OIsbyPCRandDNAhybridizationin17isolatesofShigatoxinproducing(Stx-positive)E.coliO157:H7,andcomparedwiththeirdistributionin21isolatesofStx-negativeE.coliO157and21isolatesofnon-O157entericpathogens.Fourteenof22OIswerepresentinnon-O157entericpathogensanalyzed.Eightof22OIswerefoundonlyinthe17Shigatoxin-(Stx)positiveE.coliO157:H7isolates,buttheywereabsentfromthe21Stx-negativeE.coliO157:NMandO157Hundisolatestested.Amongthe8OIs,onlyOI43orOI48wereexclusivelydetectedinStx-positiveE.coliO157:H7,absentfromneitherofStx-negativeE.coliO157andnon-O157entericpathogens,suchasSalmonella,ShigeUa,Citrobacter,Vibriocholera,enteropathogen-icE.coli(EPEC),enteroadherentE.coli(EAEC),enteroinvasiveE.coli(E1EC)andenterotoxingenicE.coli(ETEC).TheOI43andOI48are83kbinsizeandidenticalinDNAsequences,whichencodegenesforurease,telluriteresistanceandadherence.ByanalyzingtheirjunctiongeneswithPCRandDNAhybridization,wefoundthat21ChineseisolateshaveOI48only.However,for7Japanesepatientisolates,4haveOI43and3haveOI48;forAmericanisolates,2havebothofO143andOI48,2haveOI48only.ThesedataconfirmedthehighlyplasticityofthepathogenicE.coligenome.TheuniquepresenceofOI43/OI48inStx-positiveE.coli0157:H7denotesitscriticalroleinthepathogenicityspecifictothispathogen.