PDXl与PAX4双基因表达腺病毒载体构建与表达.docVIP

PDXl与PAX4双基因表达腺病毒载体的构建与表达 　　摘要： 运用pADxsi系统制备可表达人PDX1与PAX4双基因的重组5型腺病毒。从pEGFP-N1-PDX1质粒上酶切下目的基因PDX1并酶连到腺病毒穿梭质粒pShuttle-EGFP-CMV上，替换EGFP而得到pShuttle-CMV-PDX1；再将PAX4从pEGFP-N1-PAX4质粒上酶切下连到pShuttle-CMV-PDX1的多克隆酶切位点而得到穿梭质粒pShuttle-CMV-PDX1/CMV-PAX4；双酶切pShuttle-CMV-PDX1/CMV-PAX4将CMV-PDX1/CMV-PAX4转移到ADxsi骨架质粒上，得到pADxsi-CMV-PDX1/CMV-PAX4腺病毒质粒，然后在293细胞中进行包装并扩增重组腺病毒，并进行病毒滴度测定；体外感染人间充质干细胞。依据酶切、测序和PCR的结果均证明重组人PDX1与PAX4双基因表达腺病毒载体构建正确；而RT-PCR与WB结果也显示目的基因在细胞中稳定表达。应用重组技术成功构建人PDX1与PAX4双基因表达5型腺病毒载体，转录因子PDX1与PAX4在间充质干细胞内稳定表达且定位于细胞核内 关键词： 5型腺病毒；间充质干细胞（MSCs）；胰腺十二指肠同源框蛋白1（PDX1）；成对盒基因4（PAX4）；转录因子 中图分类号：Q78文献标志码：A 文章编号：1672-1098（2016）02-0080-07 Abstract：With the use of pADxsi system， the recombinant adenovirus of expressing PDX1 and PAX4 is in place.GFP in the pShuttle-GFP-CMV vector is replaced with the target gene of PDX1 obtained from the pEGFP-N1-PDX1 plasmid， resulting in the pShuttleCMV-PDX1 plasmid. With the pEGFP-N1-PAX4 plasmid cut off， PAX4 is inserted into pShuttle-CMV-PDX1 to acquire the pShuttle-CMV-PDX1/CMV-PAX4 plasmid. Afterwards， the CMV-PDX1/CMV-PAX4 fragment is transferred to the ADxsi skeleton vector to get the pADxsi-CMV-PDX1/CMV-PAX4 virus vector. Finally， the recombinant adenovirus is packaged， amplificated， and titrated in HEK293 cells for the infection of human umbilical cord mesenchymal stem cells. The structure of pADxsi-CMV-PDX1/CMV-PAX4 adenovirus expression vector is confirmed by means of a restriction analysis and PCR. RT-PCR and Western blot results have established that the target genes are persistently expressed in the infected cells.The recombinant human PDX1 and PAX4 expressing adenovirus is successfully constructed， packaged， and amplificated.The target genes can be continuously presented in mesenchymal stem cells. Key words：adenovims vector；pancreatic and duodenal homeobox factor 1；paired box gene 4；mesenchymal stem cells 糖尿病是由于胰岛β细胞功能绝对或相对缺陷，以慢性高血糖为特征的终身性代谢性疾病[1-2]。长期血糖增高，可导致大血管、微血管受损并危及心、脑、肾、周围神经、眼睛、足等，每年因糖尿病死亡者有一半以上是心脑血管所致，10%是肾病变所致；因糖