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.