NT-3基因过表达慢病毒载体的构建和包装及鉴定-论文.pdf

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1、·680·JApoplexyandNe~ousDiseases，August2014，Vol3l，No．8文章编号：1003-2754(2014)08-0680-04NT．3基因过表达慢病毒载体的构建和包装及鉴定谢柳，雷水生，赵元淑，邓镇，余涵，陈丽，胡景鑫，朱晓琴摘要：目的构建神经营养因子一3(neurotrophin-3，NT一3)基因慢病毒载体，检测其在大鼠骨髓问充质干细胞(mesenchymalstemcells，MSCs)中的表达。方法体外扩增NT一3，将NT一3全长载体GV287一GFP与扩增出的NT．3用AgeI进行酶切，

2、将NT一3全长序列克隆人GV一287一GFP，转化大肠杆菌DHSa感受态细胞，筛选出阳性克隆进行基因测序。重组GV287一EGFP质粒、pHelper1．0质粒和pHelper2．0质粒三质粒共转染至包装细胞293T，培养48h后收集细胞上清液，将病毒浓缩后在293T细胞中测定病毒滴度，并检测慢病毒载体在MSCs的转染效率。荧光显微镜观察转染是否成功，RT．PCR和Westernblot检测MSCs细胞中NT．3蛋白的表达。结果测序结果和Westernblot检测均证明NT一3慢病毒载体构建正确，且在细胞中正确表达。与辅助质粒共包装细胞

3、获得慢病毒颗粒，并成功感染MSCs细胞。包装慢病毒、浓缩病毒悬液的滴度为2×10／ml慢病毒浓缩液。结论成功构建了稳定高效表达NT-3基因的慢病毒载体。关键词：神经营养素一3；骨髓间充质干细胞；293T细胞；慢病毒载体中图分类号：R742．1文献标识码：AConstruction，packagingandidentificationoflentiviralvectorwithoverexpressiongeneNT·3XIELiu，LEIShui—sheng，ZHAOYuan—shu，eta1．(DepartmentofPhysiolo

4、gy，GuangzhouMedicalUniversity，Guangzhou510182，China)Abstract：ObjectiveConstructionofneurotrophin一3(neurotrophin一3，NT一3)genelentiviralvectoranddetectitsexpressioninratbonemarowmesenchymal(Mesenchymalstemcells，MSCs)．MethodsAmplificationNT一3geneinvitro，theNT一3full-lengthGV2

5、87一GFPvectorandamplifiedNT一3wasdigestedwithAgeI，thefull—lengthsequenceofNT-3wasclonedintoGV287-GFP，transformE．coliDHSacompetentcells，positiveclonesweresequenced．ThreeplasmidsincludingecombinantGV287一EGFPplasmid、pHelper1．0andpHelper2．0plasmidsweretransfectedintopackagingc

6、ells293T，cul—turesupematantwerecollectedafter48h，theviruswasconcentratedandtiteredin293Tceils，anddetectingLentiviralvec—tortransfectionefficiencyofBMSCsby．fluorescencemicroscopy．RT．PCRandWesternblotdetectedNT．3proteinexpress．inginBMSCscells．ResultsResultsofsequencingandW

7、esternblotresuhsprovedthatNT一3recombinantlentiviralvectorwasconstructedcorrectlyandcouldbeexpressedcorrectlyinthecel1．Lentiviralparticleswereacquiredinplasmidco—trans—fectedsecondarypackagingcellsandsuccessfullyinfectedBMSCscells．Lentiviralconcentratedvirussuspensiontite

8、red2×10’／m1．ConclusionWesuccessfullyconstructastableandeficientlentiviralvectorexpressingNT一3gene．Keywo