嗜热菌β-糖苷酶基因乳腺特异性表达载体的构建、转染及鉴定-发育生物学专业论文.docx

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嗜热菌β-糖苷酶基因乳腺特异性表达载体的构建、转染及鉴定-发育生物学专业论文.docx

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嗜热菌β-糖苷酶基因乳腺特异性表达载体的构建、转染及鉴定-发育生物学专业论文

StudyonConstructionandTransfectionofMammaryGland SpecificExpressionVectorforSsβ-glyAbstractProducingbioactiveenzymaticoflactosethroughmammaryglandbioreactoris a methodtorelieflactoseintolerance,thekeypointis theconstructionofexpressionvector inmammary.Forthepurposeof constructingmammaryglandspecificexpressional vector,theSulfolobusSolfataricus β-Glycosidases wascodonoptimizedandsynthetized basedonthecodonusefrequencyofbovine.Thebovinelactoglobulin(BLG) elementwas clonedandligatedtocompletethevector,we usetheupdreamregulationsequenceof bovineBLGtoregulateLacSgenetoexpressinmammarygland.Inordertoenhancethe screenefficiencyand safetyoftransgenicPositiveEGFPandneomycinmarkergene anchoragedbyLoxp sequence.Vectorplasmidweretransfectedintobreastcancercells inmicethroughliposome-mediatedmethod,andmonoclonalwerescreened.ThePCR integrationtestinghavebeenmadeafteranumberofpositivemonoclonalmixed, prolactin-inducedexpressionwas detectedafterRT-PCR.Thestudyresultsareas follows:Firstly,the924bpbovinebeta-lactoglobulin(BLG)gene5regulatorysequencewas amplifiedbyPCR,andconnectionwithTvector calledthePMD-19BLG,sizeofthe plasmidis3.7kb.ItwasconnectedwiththeoptimizedplasmidpUC57-Lacsandplox-EGFPplasmidafterdigestingof correspondingRestrictionenzyme,constructedthe recombinantmammaryspecificexpressionvectorPlox-EGFP-Lacs-BLG.Bovinebeta-lactoglobulingene5flankingsequencestartthethermophilicbacteriaglucosidase geneexpressioninbreast-specific,andtheneomycinresistancegeneandenhancedthe expressionofgreenfluorescentproteingenewhichisconvenienttofacilitatepositive clones.Secondly,we thawedmousebreastcancercellsin37°C waterbath,andthe determinationofofG418theminimumlethalconcentrationwasmade,then reconstructioncarrierweretransfectedintomousebreastcancercellsbyliposome-mediatedmethod.thentheintegrationof carrierdetectionandexpressionwas detectedbyPCRandRT-PCRmethod.The resultsshowedthatG418minimumlethalconcentrationofmousebreastcancer cellsis 700μg/mL,thegeneintegrationidentifiedbyPCR showedthat