基因克隆表达及功能研究1 ppt课件

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1、Genecloning,expressionandfunctionalstudy基因克隆，表达及功能研究vectorsCloningvectors:克隆载体tocloneageneinavectorExpressionvectors:表达载体toexpressagenefromavectorIntegrationvectors:整合载体tointegrateageneinagenomethroughavectorCloningvectors1Plasmidvecters2Bacteriophagev

2、ectors3Cosmids&BACs4EukaryoticvectorsCloningvectors:allowingtheexogenousDNAtobeinserted,stored,andmanipulatedmainlyatDNAlevel.expressionvectors:allowingtheexogenousDNAtobeinserted,stored,andexpressed.Containsanoriginofreplication,allowingforreplication

3、independentofhost’sgenome.ContainsSelectivemarkers:Selectionofcellscontainingaplasmidtwinantibioticresistanceblue-whitescreeningContainsamultiplecloningsite(MCS)Easytobeisolatedfromthehostcell.AplasmidvectorforcloningAmpicillinresistant?yesyesTetracycl

4、ineresistant?NoyesBXBBBXAmproriAmprTcroripBR322AmprTcrori-ScreeningbyinsertionalinactivationofaresistancegeneTwinantibioticresistancescreeningReplicaplating:transferofthecoloniesfromoneplatetoanotherusingabsorbentpadorVelvet(绒布).transferofcolonies+ampi

5、cillin+ampicillin+tetracyclinethesecolonieshavebacteriawithrecombinantplasmidBluewhitescreeningAmproripUC18(3kb)MCS(Multiplecloningsites,多克隆位点）LacpromoterlacZ’ScreeningbyinsertionalinactivationofthelacZgeneTheinsertionofaDNAfragmentinterruptstheORFofla

6、cZ’gene,resultinginnon-functionalgeneproductthatcannotdigestitssubstratex-gal.Recreatedvector:bluetransformantsRecombinantplasmidcontaininginsertedDNA:whitetransformantsRecreatedvector(noinsert)Recombinantplasmid(containinsert)backMultiplecloningsitesM

7、ultiplerestrictionsitesenabletheconvenientinsertionoftargetDNAintoavectorAmproripUC18(3kb)MCS(Multiplecloningsites,多克隆位点）LacpromoterlacZ’…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA….ThrAsnSerSerValProGlyAspProLeuGluSerThrCysArgHisAlaSer…Eco

8、RISacIKpnISmaIXmaIBamHIXbaISalIHincIIAccIPstISphILacZAplasmidvectorforgeneexpressionExpressionvectors:allowingtheexogenousDNAtobeinserted,storedandexpressed.PromoterandterminatorforRNAtranscriptionarerequired.IntactORFandribosomalbindin