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1、PDX1与NKX6.1双基因表达腺病毒载体的构建、鉴定与表达唐小龙1,2，张荣波2，汪雪峰2，张文1（1广州中医药大学第二附属医院(广东省中医院)最好保留检验科,广州510120；2安徽理工大学医学院生物工程研究所,安徽淮南232001）摘要：目的运用pADxsi系统构建带人PDX1与NKX6.1双基因表达腺病毒载体。有测序鉴定。方法采用基因克隆技术，将目的基因NKX6.1克隆至pShuttle-GFP-CMV中，得到pShuttle-GFP-CMV-NKX6.1；再将PDX1克隆至pShuttle-CMV-GFP/CMV-NKX6.1上，替换掉GFP，
2、得到pShuttle-CMV-PDX1/CMV-NKX6.1；最后将CMV-PDX1/CMV-NKX6.1从pShuttle-CMV-PDX1/CMV-NKX6.1转移到ADxsi骨架载体上，得到pADxsi-CMV-PDX1/CMV-NKX6.1，然后在293细胞中进行包装与扩增活病毒，病毒滴度测定；体外感染人胎肝间充质干细胞。结果酶切鉴定和PCR证明重组人PDX1与NKX6.1双基因表达腺病毒载体构建正确，可高效感染人胎肝间充质干细胞，并特异性地定位于细胞核内。结论应用pADxsi系统重组技术成功构建了人PDX1与NKX6.1双基因表达腺病毒载体。关
3、键词：腺病毒；胰腺十二指肠同源框1基因；NK6转录因子相关,基因座1；间充质干细胞中图法分类号：文献标识码：AConstructionandexpressionofPDX1andNKX6.1double-genemodifiedadenovirusvectorsTANGXiao-long1,2，ZHANGRong-bo2，WANGXue-feng2，ZHANGWen11ClinicalLaboratory,SecondAffiliatedHospital,GuangzhouUniversityofTraditionalChineseMedicine(Gu
4、angdongProvincialHospitalofTCM),Guangzhou510120；2ResearchSectionofBiomedicalEngineering,MedicalCollege,AnhuiUniversityofScienceandTechnology,Huainan232001,ChinaAbstract：ObjectiveConstructingthePDX1andNKX6.1double-genemodifiedadenovirusvectors,toexploretheabilityandmechanismofdiff
5、erencefromstemcelltoPancreaticbeta-cellsinducedbyPDX1andNKX6.1.MethodsThepShuttle-GFP-CMV-NKX6.1wasobtainedbythecloningoftheinterestgeneNKX6.1intopShuttle-GFP-CMV;pShuttle-CMV-PDX1/CMV-NKX6.1wasconstructedwhenGFP’sinsteadofPDX1;theCMV-PDX1/CMV-NKX6.1wastransferredfrompShuttle-CMV
6、-PDX1/CMV-NKX6.1toADxsibackboneplasmidtoconstructpADxsi-CMV-PDX1/CMV-NKX6.1,andthenthepADxsi-CMV-PDX1/CMV-NKX6.1adenovirusvectorswasamplifiedin293cells.Afterthedetectionofviraltiter,theyweretransfectedintohumanplacentalmesenchymalstemcells.ResultsThecorrectconstructionofPDX1andNK
7、X6.1double-genemodifiedadenovirusvectorwereconfirmedbyrestrictionenzymeanalysisandPCR.Therecombination genecouldinfecthumanplacentalmesenchymalstemcellsandlocatedinnucleolusspecifically.ConclusionInourstudy,weconstructedPDX1andNKX6.1double-genemodifiedadenovirusvectorsusingpADxsi
8、recombination techniquesuccessfully.Keyw
