deltex-1腺病毒载体的构建及其在大鼠bmscs中的表达

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1、deltex-1腺病毒载体的构建及其在大鼠bMSCs中的表达王荥，王延洲，徐惠成（400038重庆，第三军医大学西南医院妇产科）[摘要]目的构建deltex-1基因腺病毒载体并研究其在大鼠骨髓间充质干细胞（bonemarrowmescenchymalstemcells，bMSCs）中的表达。方法采用高保真酶PCR扩增deltex-1基因并测序，亚克隆至pAdTrack-CMV腺病毒穿梭载体后在BJ5183细菌中进行重组，挑选正确的重组子在FT293细胞中包装成病毒。以CsCl2方法纯化病毒，并进行滴度测定。将目的基因病毒感染bMSCs细胞后收集总RNA及蛋白，用PCR及Wester

2、nblot方法检测目的基因表达水平。结果①成功扩增了大小为1883bp的大鼠deltex-1，并成功构建了大小为4.5kb的同源重组腺病毒穿梭载体，滴度为1.23×1011IU/ml；②对培养的大鼠bMSCs进行流式检测，结果为CD90、CD44阳性表达，分别为99.57%和85.66%，而CD45表达阴性，阳性率为0.35%；③荧光显微镜观察deltex-1转染效率为60%～70%，RT-PCR及Westernblot检测结果显示转染后deltex-1表达明显增强，说明转染成功。.结论成功构建deltex-1基因腺病毒载体并能在bMSCs中高表达。[关键词]deltex-1基因；

3、克隆；腺病毒载体；骨髓间充质干细胞；表达[中图法分类号][文献标志码]AExpressionofdeltex-1inratbonemarrowmescenchymalstemcellsbyadenovirusvectorWangYing,WangYanzhou,XuHuicheng(DepartmentofObstetricsandGynaecology,SouthwestHospital,ThirdMilitaryMedicalUniversity,Chongqing,400038,China)[Abstract]ObjectiveToconstructanadenovirusv

4、ectorofratdeltex-1geneandtheninvestigateitsexpressioninratbonemarrowmescenchymalstemcells(bMSCs).MethodsAfterdeltex-1genewasamplifiedbyhigh-fidelityPCR,theproductwassubclonedtothepAdTrack-CMVadenovirusvectoraftersequencing,andrecombinedintheBJ5183bacterium.Thecorrectrecombinantwerepickedandthe

5、npackagedtovirusinFT293cells.ThentheviruswaspurifiedwithCsCl2anditstiterwasdetermined.Atlast,theexpressionofdeltex-1genewasdeterminedbydetectingthecontentsoftotalRNAandproteinofthebMSCswiththetargetgenebyPCRandWesternblottingrespectively.ResultsRatdeltex-1genefragmentof1883bpwassuccessfullyamp

6、lify,andconstructedinthereconstitutedadenovirusshuttlevectorof4.5kb,andthetiterofthevectorwas1.23×1011IU/ml.Flowcytometricanalysisshowedcell-surfaceantigensforratbMSCswereCD9099.57%,CD4485.66%andCD450.35%.Theefficiencyoftransfectionwas60%to70%underfluorescentmicroscope.RT-PCRandWesternblotting

7、suggestedthattheexpressionofdeltex-1oftransfectedMSCswasobviouslyhigherthancontrols.Alloftheserevealthatthetransfectionofdeltex-1intoratbMSCswassuccessful.ConclusionTheadenovirusvectorofdeltex-1issuccessfullyconstructedandthebMSCsinfect