携带NMU2R shRNA重组腺相关病毒载体的构建及其评价

Construction and appraisal of recombinant adeno-associated virus vector for expression shRNA targeting NMU2R mRNA

构建稳定表达 NMU2R shRNA的重组腺相关病毒载体,制备并纯化靶向性沉默 NMU2R 表达的高滴度重组腺相关病毒。首先设计合成 NMU2R shRNA,退火形成双链与pAAV-ZsGreen-shRNA质粒 Bam H I和 Hin d III双酶切产物相连接构建形成质粒pAAV-ZsGreen-r NMU2R shRNA,经双酶切和测序鉴定证实,重组质粒克隆正确,将重组质粒转染到AAV-293包装细胞中包装成腺相关病毒载体,成功制备重组腺相关病毒rAAV5-ZsGreen-r NMU2R shRNA,经测定纯化所得rAAV5病毒滴度为1×10^12 vg/mL。最后将rAAV5-ZsGreen-r NMU2R shRNA感染大鼠嗜铬细胞瘤细胞PC12,检测 NMU2R shRNA基因沉默效果,利用空病毒载体pAAV-ZsGreen-shRNA作对照,Real-time PCR及Western Blot方法测得 NMU2R mRNA及蛋白表达水平与对照组相比显著下降。综上,成功构建了高滴度携带 NMU2R shRNA重组腺相关病毒载体rAAV5-ZsGreen-r NMU2R shRNA。

To construct the recombinant adeno-associated virus vector carrying neuromedin U 2 receptor ( NMU2R ) short hairpin RNA (shRNA) for preparation of high-titer viruses, hairpin RNA was designed to target NMU2R rat mRNA, and was synthesized and cloned into pAAV-ZsGreen-shRNA plasmid which was double digested by Bam H I and Hin d III. The presence of the target sequence in the plasmid pAAV-ZsGreen-r NMU2R shRNA was confirmed by enzyme digestion, PCR and DNA sequencing analysis. And then the plasmid was transfected into AAV 293 cells. AAV 293 cells expression NMU2R shRNA were obtained and subsequently infected AAV packaging system to package the rAAV5-ZsGreen-r NMU2R shRNA. After purification, the functional and infectious virus was obtained and the titer of virus was 1×10^12 vg/mL. Real-time PCR and western blot methods were used to detect the expression of NMU2R after infection with PC12 cells, and the empty viral vector pAAV-ZsGreen-shRNA was used as control. The results indicated that the expression of NMU2R mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the control. In summary, a high-titer recombinant adeno-associated virus-5 vector carrying NMU2R shRNA has been constructed successfully.