利用同尾酶技术构建pet15b

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1、利用同尾酶技术构建pET15b作者：姚玲玲，王家宁，黄永章，郭凌郧［摘要］目的:-PEP-1-CAT重组质粒，为制备PEP-1-CAT融合蛋白进而为防治心肌缺血再灌注损伤奠定基础。方法：设计并合成两端分别带SalI和BglII酶切位点的CAT引物，用PCR方法以pZeoSV2(+)-CAT质粒为模板，特异性扩增CAT全长cDNA。将扩增的CATcDNA与dATP反应，利用TaqDNA聚合酶具有的非模板依赖性末端转移酶活性在CATcDNA的3'末端加"A"并纯化，然后应用TA克隆策略将纯化后的CATcDNA插入至

2、中间载体pGEM-TEasyVector中，经PCR、限制性酶切分析鉴定重组子pGEM-T-CAT。人工合成编码PEP-1的双链寡核苷酸，两端分别引入NdeI和XhoI酶切位点，将其插入pET15b中得到重组质粒pET15b-PEP-l，然后酶切鉴定。pGEM-T-CAT和pET15b-PEP-1分别经SalI-BglII和XhoI-BamHI双酶切，利用同尾酶能酶切产生相同黏性末端的特性，将回收得到的CATcDNA片段与pET15b-PEP-l相连，构建出重组质粒pET15b-PEP-l-CAT，经PCR及酶

3、切鉴定，并通过核苷酸序列测序证实。结果：获得pET15b-PEP-l-CAT重组子，经测序分析证实，克隆入pET15b的PEP-1序列与设计合成的PEP-1序列一致，CAT序列与GeneBank中登录号为“AY028632”的CATcDNA序列一致。结论:pET15b-PEP-l-CAT的成功构建为产生融合蛋白Histag-PEP-1-CAT进而为防治心肌缺血再灌注损伤奠定了基础。［关键词］同尾酶；过氧化氢酶；TA克隆；DNA重组；PEP-1肽Abstract：ObjectiveToconstructthere

4、combinantplasmidpET15b-PEP-1-CATwithisocaudamertechniqueandprovideavectorexpressingPEP-l-CATfusionprotEinforthepreventionandtherapyofmyocardialischemia-reprefusionUsingpfuDNApolymerase,thefull-lengthhumancatalasecDNAwasspecificallyamplifiedviaPCRfrompZeoSV2(

5、+)-CATplasmid.ThePCRproductwasaddednA,rbyusingthetemplate-independentterminaltransferaseactivityofTaqDNApo1ymerase.ThepurifiedproductsofCATcDNAwhose3!endwasaddednA"were1igatedwithpGEM-TEasyvector,andweretransformedintoDH5a•Thecorrectrecombinantwasidentifiedb

6、yPCRandSalI-BglIIdigestionandnamedafteroligoncleotidesweresynthesizedandannealedtogenerateadouble-strandedoligonucleotideencodingthePEP-1PEP-lpeptidesequenCATwerefurtherdigestedbyXhoI-BamHIandSall-BglII,respectively.ThepurifiedlinearfragmentofpETcewasdirectl

7、yligatgestedrecombinantpdbydouble-enzymedirpET15b-PEP-l.pETledintoNdel-Xhol-dilasmidwasidentifiegestedandnamedafte5b-PEP-landpGEM-T-15b-PEP-landthepurifiedCATcDNAfragmentwereligatedbyusidamerswhichhavedifequencesbutproduceends.TheplasmidwitwasselectedusingaX

8、ysisandthenanalyseItsTherecombinantpthatthePEP-landtheceofpET15b-PEP-l-CconsistentwithdesidthehumancatalasecanknAY028632n,resp-PEP-1-CATofprokarsmidwasconstruetedusionConstructionngtwopairsofiso