Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

(整期优先)网络出版时间:2004-01-11
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Tocloneandconstructtherecombinantplasmidcontainingthemajoroutermembraneprotein(MOMP)geneofChlamydiatrachomatis(C.trachomatis)andtoexpressthefusionproteininE.coliBL21,theMOMPgenewasamphfiedbypolymerasechainreaction(PCR)fromgenomeofC.trachomatisserovarD.ThefragmentwasclonedintotheprokaryoticexpressionvectorpET-22b(+)afterdigestionwithBamHⅠandNotⅠandtransformedintoE.coliXL1-Blue.RecombinantswereselectedbyenzymedigestionandsequencingandtherecombinantplasmidwithMOMPgenewasthentransformedintoE.coliBL21withIPTGtoexpressthetargetgene.TheexpressionrecombinantproteinswerepurifiedbyNi-NTAaffinitychromatography,andidentifiedbySDS-PAGEandWesternblot.Itwasfoundthata1.2kbMOMPgenewasisolated.TheDNAsequenceofMOMPwasfoundtobejustthesameasthesequencepublishedbyGenBank.ArecombinantplasmidcontainingMOMPgenewasconstructedtoexpressthefusionproteinsinE.coli.SDS-PAGEanalysisshowedthattherelativemolecularweightoftherecombinantproteinwasabout47kDathatwasconsistentwiththetheoreticalpredictedvalue,andthespecificityoftheexpressedproteinwasconformedbyWesternblot.ItconcludedthattheMOMPgenecouldbeexpressedintheprokaryoticsystem,bywhichitprovidedthefoundationforthefuturestudiesonthebiologicalactivitiesofC.trachomatisandforthedevelopmentofvaccineagainstthispathogen.