摘要
目的构建含有小鼠过氧化物酶增殖激活受体δ(peroxisom e proliferate activated receptor delta,PPARδ)基因的重组腺病毒表达载体。方法通过KpnⅠ和EcoRⅤ双酶切,从pCDNA3.0-mPPARδ载体中得到含有小鼠PPARδ全长编码序列的片段,将mPPARδ与pAdtrack-CMV相连接并用Pm eⅠ线性化后,转化含有pAdeasy骨架质粒的细菌B J5183,在细菌内同源重组后得到pAd-mPPARδ质粒。pAd-mPPARδ经PacⅠ线性化后用L ipofectam ineTM2000转染HEK293细胞,包装得到含PPARδ基因的病毒重组子,将病毒重组子在HEK293细胞中扩增后,反复冻融得到滴度较高的含PPARδ的病毒液。将收集的病毒液感染INS-1细胞,绿色荧光观察GFP和W estern b lot检测PPARδ蛋白的表达。结果成功构建了含有小鼠PPARδ基因的重组腺病毒载体,病毒滴度为1.5×1010pfu/m。l重组腺病毒感染后可使INS-1细胞PPARδ蛋白表达明显增加。结论该重组腺病毒载体的构建为研究PPARδ基因在胰岛细胞中生物学功能提供了一定的工作基础。
Objective To construct the recombinant adenovirus vector containing mouse PPARδ,and infect the INS-1 cells. Methods The pCDNA3.0-mPPAR5 plasmid was digested with Kpn Ⅰ and EcoR V to obtain full-length coding sequence of mPPARδ, then the mPPARδ cDNA was ligated to the pAdtraek-CMV vector. The plasmid of pAdTrack-mPPARδ was linearized with Pine-Ⅰ , and the fragment was reclaimed and transformed into BJ5183 which contains pAdeasy. After being screened, the extracted plasmid of positive bacteria linearized with Pac Ⅰ was transfected into HEK293 cells with lipofectamine^TM 2000 and was identified by the green fluorescence protein （GFP）. The harvested virus was amplified in HEK293 cells and infected INS-1 cells. After infection, the expression of PPAR5 was proved by GFP expression and Western blotting. Results The recombi nant adenovirus vector containing mouse PPARδ was constructed successfully and the titer was 1.5 × 10^10 pfu/ ml. The PPAR5 expression in INS-1 cells infected by the virus was much higher than the controls. Conclusion The constructed recombinant adenovirus vector containing mouse PPARδ provides a potent tool to investigate its biological function in pancreatic islets and other tissues.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第1期68-70,共3页
Journal of Third Military Medical University
