人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定栗炳南，李卫东，林俊堂，丰慧根新乡医学院生命科学技术学院，河南省新乡市453003Constructionandidentificationofbicistroniceukaryoticexpressionvectorofhumanbrain-derivedneurotrophicfactorandneurotrophine-3LiBing-nan,LiWei-dong,LinJun-tang,FengHui-genDepartmentofLifeSciencesandTechnology,XinxiangMedicalUniversity,Xinxiang453003,HenanProvince,China摘要背景：脑源性神经营养因子(brain-derivedneurotrophicfactor，BDNF)和神经营养素3(Neurotrophines-3，NT-3)在细胞分化过程中有重要作用。病毒载体临床应用存在安全隐患，利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。目的：构建双基因共表达载体pIRES2-BDNF-NT-3并对其进行鉴定。方法:BDNF和NT-3基因核心序列是通过直接PCR的方法从外周血单个核细胞的基因组DNA中获取。然后将BDNF的cDNA片段插入到pIRES2-EGFP多克隆位点构建pIRES2-BDNF-EGFP载体，随后将NT-3cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中，最后构建成为含有内部核糖体进入位点(IRES)的pIRES2-BDNF-NT-3双基因共表达载体。实验通过双酶切和DNA测序方法对其鉴定，将重组的双基因共表达载体感染HEK293细胞，利用RT-PCR与Western-blot方法检测双基因的表达。结果与结论：DNA测序显示，提取的BDNF和NT-3均与基因库报道序列一致。构建的pIRES2-BDNF-NT-3双基因共表达载体经EcoRⅠ/BamHⅠ切出BDNF条带，经BamHⅠ/NotⅠ双酶切后切出IRES-NT-3片段，经EcoRⅠ/NotⅠ双酶切后切出BDNF-IRES-NT-3片段。RT-PCR与Western-blot方法检测显示，此载体转染后的HEK293细胞均能表达BDNF和NT-3mRNA和蛋白。结果证实，实验成功采用IRES序列构建了能分别表达的BDNF和NT-3双基因真核表达载体。中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：关键词：组织构建；组织工程；脑源性神经营养因子；神经营养素3；真核双表达载体；转染；双PCR；Abstract：BACKGROUND:Humanbrain-derivedneurotrophicfactor(BDNF)andneurotrophine-3(NT-3)areessentialgenesforcelldifferentiation.Viralvectorhasbeenusednumerouslyinclinicalpractice,butthesecurityisthemostimportantproblem.Eukaryoticexpressingvectorisawaytosolvethisquestion.OBJECTIVE:ToconstructandidentifypIRES2-BDNF-NT-3bicistroniceukaryoticexpressionvector.METHODS:BDNFandNT-3geneswereobtainedfromthegenomicDNAofhuman---本文来源于网络，仅供参考，勿照抄，如有侵权请联系删除---peripheralbloodmononuclearcellsbyPCR.TheBDNFcDNAfragmentwasinsertedintothemultiplecloningsitesofpIRES2-EGFP,togeneratethebicistroniceukaryoticexpressionplasmidpIRES2-BDNF-EGFP.ThenNT-3cDNAfragmentwasclonedintothepIRES2-BDNF-EGFP,insteadofEGFP,tocreateplasmidpIRES2-BDNF-NT-3.SubsequentlypIRES2-BDNF-NT-3wasusedtotransfectHEK293cells,andRT-PCRandwesternblotanalysiswereappliedtotesttheco-expressionofdoublegenes.RESULTSANDCONCLUSION:TheDNAsequencinganalysisdemonstratedthattheBDNFandNT-3wereexactlyconsistentwiththesequencerecordedintheGenBank.Enzymedigestionanalysisindicatedthat,intheconstructedbicistroniceukaryoticexpressionvectorpIRES2-BDNF-NT-3,BDNFbandwasobtainedbyEcoRI/BamHIdigestion,IRES-NT-3fragmentwasobtainedbyBamHI/NotIdigestion,andBDNF-IRES-NT-3wasobtainedbyEcoRI/NotIdigestion.RT-PCRandwesternblotanalysisshowedthat,aftertheHEK293cellsweretransfectedwithpIRES2-BDNF-NT-3,doublegenewasexpressedatthemRNAandproteinlevel.Experimentalfindingssuggestthat,bicistroniceukaryoticexpressionvectorofBDNFandNT-3genescanbesuccessfullyconstructedusingIRESsequence.中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成...
