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ID:19515522
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页数:30页
时间:2018-10-03
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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tointegrateageneinagenomethroughavectorCloningvectors1Plasmidvecters2Bacteriophagevectors3Cosmids&BACs4Eukary
2、oticvectorsCloningvectors:allowingtheexogenousDNAtobeinserted,stored,andmanipulatedmainlyatDNAlevel.expressionvectors:allowingtheexogenousDNAtobeinserted,stored,andexpressed.Containsanoriginofreplication,allowingforreplicationindependentofhost’sgenome.ContainsSelectivemarkers:S
3、electionofcellscontainingaplasmidtwinantibioticresistanceblue-whitescreeningContainsamultiplecloningsite(MCS)Easytobeisolatedfromthehostcell.AplasmidvectorforcloningAmpicillinresistant?yesyesTetracyclineresistant?NoyesBXBBBXAmproriAmprTcroripBR322AmprTcrori-Screeningbyinsertion
4、alinactivationofaresistancegeneTwinantibioticresistancescreeningReplicaplating:transferofthecoloniesfromoneplatetoanotherusingabsorbentpadorVelvet(绒布).transferofcolonies+ampicillin+ampicillin+tetracyclinethesecolonieshavebacteriawithrecombinantplasmidBluewhitescreeningAmproripU
5、C18(3kb)MCS(Multiplecloningsites,多克隆位点)LacpromoterlacZ’ScreeningbyinsertionalinactivationofthelacZgeneTheinsertionofaDNAfragmentinterruptstheORFoflacZ’gene,resultinginnon-functionalgeneproductthatcannotdigestitssubstratex-gal.Recreatedvector:bluetransformantsRecombinantplasmidc
6、ontaininginsertedDNA:whitetransformantsRecreatedvector(noinsert)Recombinantplasmid(containinsert)backMultiplecloningsitesMultiplerestrictionsitesenabletheconvenientinsertionoftargetDNAintoavectorAmproripUC18(3kb)MCS(Multiplecloningsites,多克隆位点)LacpromoterlacZ’…ACGAATTCGAGCTCGGTA
7、CCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA….ThrAsnSerSerValProGlyAspProLeuGluSerThrCysArgHisAlaSer…EcoRISacIKpnISmaIXmaIBamHIXbaISalIHincIIAccIPstISphILacZAplasmidvectorforgeneexpressionExpressionvectors:allowingtheexogenousDNAtobeinserted,storedandexpressed.PromoterandterminatorforR
8、NAtranscriptionarerequired.IntactORFandribosomalbindin
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