tnfαtumstatin融合基因腺病毒载体的构建及其在人脐带间质干细胞中的表达

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1、TNFαTumstatin融合基因腺病毒载体的构建及其在人脐带间质干细胞中的表达【摘要】构建携带绿色荧光蛋白的TNFαTumstatin融合基因腺病毒表达载体，转染人脐带间质干细胞(humanumbilicalcordmesenchymalstemcells,hUCMSCs)。方法：设计含有GMCSF信号肽序列和BglⅡ及HindⅢ酶切位点的引物，PCR扩增TNFαTumstatin，将扩增产物亚克隆到穿梭质粒pAdTrackCMV上，重组穿梭质粒经PmeⅠ线性化后转化含有腺病毒骨架质粒pAdEasy1的BJ5183中同源重组。筛选获

2、得含有融合基因的重组腺病毒质粒，酶切鉴定并测序。重组病毒质粒用PacⅠ酶切线性化后转染293A细胞，经过包装、扩增后感染hUCMSCs并检测细胞内融合基因的表达。结果：重组腺病毒质粒经PCR和PacⅠ酶切鉴定，证实含有TNFαTumstatin融合基因，测序结果和设计片段的序列一致。重组腺病毒感染的hUCMSCs表达绿色荧光蛋白和融合基因。结论：成功构建了TNFαTumstatin融合基因的腺病毒表达载体，能高效率感染hUCMSCs，为进一步研究融合基因修饰的hUCMSCs抗肿瘤效应奠定了基础。【关键词】腺病毒载体融合基因同源重组细胞转染［A

3、bstract］Objective:Toconstructgreenfluorescenceprotein(GFP)labeledrecombinantadenovirusvectorcarryingTNFαTumstatinfusiongeneandtransfectitintohUCMSCs.12Methods：TheamplificationproductsofTNFαTumstatinbyPCRwithapairofprimerswithBglⅡandHindⅢrestrictionendonucleasesitesandsignal

4、peptidesequenceofGMCSFweresubclonedintoshuttleplasmidpAdtrackCMVafterdigestingwithBglⅡandHindⅢ.Theresultantplasmid,afterlinearizedbydigestingwithrestrictionendonucleasePmeⅠ,wastransformedintoE.coli.BJ5183thathadbeentransformedbyadenoviralbackboneplasmidpAdEasy1.Recombinant

5、plasmidwereobtainedbyalternationofkanamycinandthenconfirmedbyPCRandrestrictionendonucleaseanalysis.Theadenoviruswaspackagedandpropagatedinhumanembryonalkidneycells(293Acells)afterbeinglinearizedbydigestingwithrestrictionendonucleasePacⅠandthenwastransfectedintohUCMSCs.Fusiong

6、eneexpressionininfectedhUCMSCswasdetectedbyRTPCR.Results：TheresultsofPCRandrestrictionendonucleaseassayindicatedthattargetgenewasinsertedintorecombinantadenovirusvectorsuccessfully.Thesequenceoffusiongenewasthesameasthatofdesignedfragments.hUCMSCsinfectedbytherecombinantaden

7、ovirusexpressedGFPandfusiongenewhichcouldbedemonstratedbyfluorescencemicroscopeandRTPCRrespectively.Conclusion：RecombinantadenovirusvectorcontainingTNFαTumstatinhasbeenconstructedsuccessfully12andcouldtransfectedhUCMSCsefficiently,whichlaidafoundationforfurtherinvestigation

8、ofantitumoreffectofhUCMSCsmodifiedwithTNFαTumstatin.［Keywords］aden