编码大鼠Nogo受体mRNA的shRNA重组腺病毒载体的构建和鉴定

Construction and identification of recombinant adenovirus expressing the shRNA of rat Nogo-66 receptor

目的:构建Nogo受体(NgR)特异性shRNA重组腺病毒载体,为应用基因沉默技术从转录后水平进行缺血性脑损伤的基因治疗研究奠定基础.方法:将前期构建的大鼠Nogo受体mRNA的shRNA特异性真核表达载体pGenesil-1-shRNA的表达启动子U6连同shRNA亚克隆至穿梭质粒pAdTrack,酶切及DNA测序鉴定后,将含NgR基因的重组穿梭质粒pAdTrack-U6-shRNA经PmeI线性化后转化入pAdEasy-1感受态细菌.将pAd-U6-shRNA质粒经PacI线性化后转染293细胞,包装重组腺病毒Adeno-U6-shRNA,并进行PCR鉴定、PCR产物测序及病毒滴度测定.结果:结果证实pAdTrack-U6-shRNA及pAd-U6-shRNA质粒构建正确,收获病毒后的PCR及DNA测序结果证明Adeno-U6-shRNA包被成功.结论:成功构建了大鼠Nogo受体mRNA的shRNA重组腺病毒载体Adeno-U6-shRNA,将为应用基因沉默技术研究NgR在缺血性脑损伤后神经再生中的作用奠定基础.

AIM： To construct the recombinant adenovirus vector of shRNA targeted to the rat Nogo-66 receptor gene for the therapy of ischemic brain injury in post-transcriptional regulation. METHODS： The used pGenesil-1-shRNA was construsted and identified in previous experiment. The U6 and shRNA of pGenesil-1-shRNA were subcloned to pAdTrack. The product pAdTrack-U6-shRNA was linearized by Pme I to mediate homologous recombination with pAdEasy-1 in pAdEasy-1 host bacteria. The positive clone was identified by enzyme digestion, PCR analysis and DNA sequence analysis. After linearized by Pac I, the recombinant adenovirus DNA pAd-U6-shRNA was transfected into 293 cells for packaging and amplification of Adeno-U6-shRNA. Adeno-U6-shRNA was further identified by PCR analysis and DNA sequence analysis. RESULTS： PCR analysis, enzyme digestion and DNA sequence analysis proved the pAdTrack-U6-shRNA and the pAd-U6-shRNA bad been successfully constructed. After being packaged in 293 cells, the recombinant adenovirus Adeno-U6-shRNA was identified by PCR analysis and DNA sequence analysis. CONCLUSION： We have successfully constructed recombinant adenovirus Adeno-U6-shRNA targeted against the rat Nogo-66 receptor gene. It will be helpful to use RNAi in the research of the role of NgR in neural regeneration after cerebral ischemic injury.