靶向大鼠TGF-β1基因shRN A真核表达载体的构建及鉴定.pdf

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1、生物医学工程研究2014JoumaofBiomedicalEngineeringResearchuL．●33(2}：。93一9J7，oamal。靶向大鼠TGF—l31基因shRNA真核表达载体的构建及鉴定米都泓莲，张千，邓存良△(泸州医学院附属医院感染病科，泸州646000)摘要：构建并鉴定靶向大鼠TGF一131基因的短发夹RNA(shorthairpinRNA，shRNA)真核表达载体，为探索肝纤维化的基因治疗提供有力工具。根据大鼠TGF一131基因的mRNA序列设计并合成2条编码shR．NA的特异性寡核苷酸片段，另选通用阴性对照序列HK，将上述片段退火后分别克隆入线性化质粒载体PGe

2、nesil一1．1中，构建出含靶基因片段的shRNA真核表达载体PGenesil—TGF一131一A、PGenesil—TGF—B1一B及PGenesil—HK，行酶切鉴定及测序分析，并采用脂质体介导法体外转染Hela细胞，荧光显微镜观察转染结果。经酶切和测序鉴定证实，构建的shRNA成功插入到载体，重组质粒的插入序列与设计的靶基因片段完全一致，3种重组质粒均能转染入Hela细胞表达绿色荧光。成功构建出靶向大鼠TGF—Bl基因的shRNA真核表达载体，为后续研究抗肝纤维化的基因药物奠定了实验基础。关键词：肝纤维化；TGF一131；RNA干扰；质粒；基因重组中图分类号：R318文献标识码：

3、A文章编号：1672-6278(2014)02-0093-05ConstructionandIdentificationofEukaryoticExpressionPlasmidsContainingShortHairpinRNATargetingatRatTGF—I3}1geneDUHonglian，ZHANGqian，DENGCunliang(InfectionDepartmentofLuzhouMedicalCollege，Luzhou646000，China)Abstract：Toconstructandidentifyeukaryoticexpressionplasmidsco

4、ntainingshorthairpinRNA(shRNA)thattargetatratTGF一131geneandtoprovideapowerfultoolforexploringapromisinggenetherapywayofhepaticfibrosis．TwopairsofshRNAsthattargetatratTGF—B1geneweredesigned，andno—isogenygenesegmentHKwasselectedasanegativecontro1．Afterannealed，theinsertswereseparatelyligatedintothe

5、lineafizedpGenesil一1．1plasmids．TheeukaryoticexpressionplasmidspGenesil—TGF一81一A，pGenesil—TGF—B1一BandpGenesil—HKwereconstructed，andthenidentifiedusingrestrictionenzymeidentificationandsequencinganalysis．ThreerecombinantplasmidsweretransfectedintoHelacellsaccordingtoLipofectamine，andEGFPexpressions

6、wereobservedunderfluorescencemicroscopeafter48h．Byrestrictionenzymeidentificationandsequencing，theDNAfragmentswereinsertedeorrranectlyinpGenesil一1．1vectorsasdesigned，respectively．ThreerecombinantplasmidsweretransfectedintoHelacells．Wesuccessfullycon—struettwoeukaryoticexpressionrecombinantplasmid

7、spGenesil一1．1一shRNAtargetedtoratTGF一131gene。anditwilllaythefoun—dationforfurtherresearchongenetreatmentofliverfibrosis．Keywords：Hepaticfibrosis；TGF一131；RNAinterference；Plasmid；Generecombianttrix，ECM)在肝内过度沉积为特点，是各种慢性肝1引