长链非编码RNA0610009 E02 Rik／Notch1腺病毒载体构建及其在原代肝细胞中的表达

Construction and Identification of Mouse IncRNA-O61OOO9EO2Rik Ad- enovirus Vector

目的构建长链非编码RNA-0610009 E02 Rik（长链非编码RNA-0610009 E02 Rik，简称Rik）重组腺病毒载体，制备一定滴度含Rik基因的重组腺病毒，为后续研究Rik的生物学功能提供实验工具。方法①应用分子克隆技术以Rik的cDNA为模板，PCR扩增，酶切。将酶切后的目的基因连接到腺病毒穿梭质粒GV315上，产生0610009E02Rik腺病毒表达载体，连接好的产物转化感受态大肠埃希菌进行克隆，对阳性克隆进行酶切鉴定并测序；②重组腺病毒穿梭质粒和辅助包装质粒pBHG在人胚胎肾293细胞中包装、扩增、纯化并测定病毒滴度；③将构建好的腺病毒感染原代肝细胞．Real．timePCR检测原代肝细胞内Rik的相对表达水平。结果①成功构建Rik重组腺病毒穿梭载体；②重组病毒经扩增、纯化，最终滴度测定约为2×10^10PFU／ml：③Real-timePCR检测过表达组肝细胞内Rik的表达量约为对照组的2440＋0．36倍（P〈0．05），差异有统计学意义。结论构建含Rik基因的腺病毒载体，在小鼠原代肝细胞内成功实现了Rik基因的表达上调，为后续开展有关Rik生物学功能及分子机制研究奠定基础。

Objective To construct and identify the mouse lncRNA-0610009E02Rik vector for further studies of its role in hepatocytes injury/repair and molecular mechanism. Methods Rik cD- NA were cloned into adenovirus shuttle vector GV315 and then the constructed shuttle plasmid and packaging plasmid pBHG were co-transfected into HEK 293 cellsfor assembly of recombination ade- novirus, amplification, ultracentrifugation. Gradient dilution method was used to detect the viral titer in HEK 293 cells. The primary hepatocytes were infected by recombination adenovirus in different multiplicity of infection and real-time PCR was used to test the level of 2Rik mRNA in hepato- cytes. Results ① The Rik gene adenovirus shuttle plasmid was successfully constructed. ② After amplification and purification, the virus titers were up to 2×10^10 PFU/ml. ③ Th Real-time PCR showed that expression of 0610009EO2Rik IncRNA were 2240 ＋ 0. 36 folds versus Control groups （P 〈0. 05 ） . Conclusion We constructed the overexpression of 0610009 EO2Rik adenovirus vector successfully, which could up-regulate transcription of 0610009 EO2Rik lncRNA in the primary hepatocytes in mice.