hhgf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、hHGF腺病毒载体的构建及其在人脐带间质干细胞中的表达作者：陈圆，李汉华，赵亚娟，吴晓丹，王梅，许晓蒙，谢颖，许文荣【摘要】　　目的：构建人重组肝细胞生长因子(humanhepatocytegrowthfactor，hHGF)基因腺病毒载体(AdHGF),转染人脐带间质干细胞（humanumbilicalcordmesenchymalstemcells,hucMSCs），为转基因治疗提供实验基础。方法：设计含有SalⅠ及XbaⅠ酶切位点的引物，PCR扩增hHGF，将扩增产物克隆到带有绿色荧光蛋白（greenfluorescenceprotein,GFP)标记的pAdTrackCMV穿梭

2、质粒上。重组穿梭质粒经PmeⅠ线性化后，与腺病毒骨架质粒pAdEasy1在BJ5183中同源重组，筛选获得含有hHGF的重组腺病毒质粒，PCR、酶切鉴定并测序。重组病毒质粒用PacⅠ酶切线性化，脂质体转染293A细胞，经3轮扩增，制备高效表达的AdGFP/HGF，并转染hucMSCs。结果：重组腺病毒质粒经PCR和SalⅠ，XbaⅠ酶切鉴定,证实含有HGF基因,测序结果和设计片段的序列一致。荧光显微镜下发现293A细胞发光率几乎为100%，感染的hucMSCs亦可表达绿色荧光蛋白。结论:成功构建了hHGF腺病毒表达载体，并获得高滴度的病毒,能高效感染hucMSCs,为后续研究奠定了基础。

3、【关键词】肝细胞生长因子；人脐带间质干细胞；腺病毒载体14　　［Abstract］Objective:Toconstructgreenfluorescenceprotein(GFP)labeledrecombinantadenovirusvectorcarryinghumanhepatocytegrowthfactor(hHGF)geneandtransfectitintohucMSCs.Methods：TheamplificationproductsofHGFbyPCRwithapairofprimerswithSalⅠandXbaⅠrestrictionendonucleasesite

4、swassubclonedintoshuttleplasmidpAdtrackCMV.Theresultantplasmid,afterlinearizedbydigestingwithrestrictionendonucleasePmeⅠ,wastransformedintoE.coliBJ5183whichhadbeentransformedbyadenoviralbackboneplasmidpAdEasy1.RecombinantplasmidwasobtainedandthenconfirmedbyPCRandrestrictionendonucleaseanalysis.T

5、headenoviruswaspackagedandpropagatedinhumanembryonalkidneycells(293Acells)afterbeinglinearizedbydigestingwithrestrictionendonucleasePacⅠ.Afterthreeamplified,highlyexpressionofAdGFP/HGFhadbeenprepared,thentransfectitintohucMSCs.Results:TheresultsofPCRandrestrictionendonucleaseassayindicatedthattarg

6、etgenewasinsertedintorecombinantadenovirusvectorsuccessfully.Thesequenceoffusiongenewasthesameasthatofdesignedfragments.293Acellswerefound100%luminanceunderfluorescencelightmicroscopy.ModifiedhucMSCscouldexpressGFP.Conclusion:Recombinantadenovirus14vectorcontaininghHGFhasbeenconstructedsuccessfull

7、yandobtainedhighlyefficientvirus,andcouldtransfecthucMSCsefficientlywhichlaidafoundationforfurtherinvestigation.　　［Keywords］hepatocytegrowthfactor；humanumbilicalcordmesenchymalstemcells；adenovirusvector　　肝细胞生长因子（