NOV基因真核表达载体的构建及在大鼠真皮多能干细胞中的表达

Construction of eukaryotic expression vectors of nephroblastoma overexpression gene and expression in rat dermal multipotent stem cells

目的构建肾母细胞瘤过度表达（NOV）基因真核表达载体并检测其在真皮多能干细胞中的表达。方法利用逆转录-多聚酶链反应（RT-PCR）方法，以新生大鼠大脑组织总RNA为模板，扩增出1l178bp的NOV基因的cDNA编码区序列，然后用Hind Ⅲ和BamH Ⅰ双酶切后定向克隆入真核表达载体pEGFP-N1质粒中，用脂质体法将重组质粒转染入大鼠真皮多能干细胞中，荧光显微镜观察转染产物．RT—PCR法检测转染细胞中NOV基因表达。结果NOV基因cDNA正确克隆到真核表达载体pEGFP-N1质粒中；重组质粒体外转染入大鼠真皮多能干细胞后，可见转染细胞有绿色荧光表达，转染细胞中检测到NOV基因。结论构建成功NOV基因重组质粒，并能在大鼠真皮多能干细胞中稳定表达，为NOV基因及真皮多能干细胞的作用研究提供了有利的分子工具。

Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma over-expression gene （NOV） and detect its expression in dermal multipotent stem cells （DMSCs）. Methods A 1 178 bp eDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pEGFP-N1. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes Hind llI and BamH I. The recombinant plasmid was transfected into DMSCs with liposome. The expression of NOV gene was detected by RT-PCR. Results Eukaryotic expression vectors containing 1 178-bp coding region of NOV gene were successfully constructed. DMSCs transfected with the recombinant plasmid expressed NOV gene. Conclusions That eukaryotic expression vector containing coding region of NOV gene is constructed and expressed in DMSCs can provide a strong molecular tool for the studies on the NOV gene and DMSCs.