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医学论文范文:编码大鼠Nogo受体mRNA的shRNA重组腺病毒载体的构建和鉴定
【摘要】 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pGenesil1shRNA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pAdTrackU6shRNA经PmeI线性化后转化入pAdEasy1感受态细菌. 将pAdU6shRNA质粒经PacI线性化后转染293细胞，包装重组腺病毒AdenoU6shRNA，并进行PCR鉴定、PCR产物测序及病毒滴度测定. 结果: 结果证实pAdTrackU6shRNA及pAdU6shRNA质粒构建正确，收获病毒后的PCR及DNA测序结果证明AdenoU6shRNA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AdenoU6shRNA，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.
【关键词】 Nogo受体；RNA干扰；腺病毒
Construction and identification of recombinant adenovirus expressing the shRNA of rat Nogo66 receptor
ZHANG QinLi1, QIN XinYue1, YIN Cheng1, PENG Yan2
1Department of Neurology, First Affiliated Hospital, 2Department of Histology and Embryology, Chongqing Medical University, Chongqing 400016, China医.学.全.在.线www.lindalemus.com
【Abstract】 AIM: To construct the recombinant adenovirus vector of shRNA targeted to the rat Nogo66 receptor gene for the therapy of ischemic brain injury in posttranscriptional regulation. METHODS: The used pGenesil1shRNA was construsted and identified in previous experiment. The U6 and shRNA of pGenesil1shRNA were subcloned to pAdTrack. The product pAdTrackU6shRNA was linearized by Pme I to mediate homologous recombination with pAdEasy1 in pAdEasy1 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 pAdU6shRNA was transfected into 293 cells for packaging and amplification of AdenoU6shRNA. AdenoU6shRNA was further identified by PCR analysis and DNA sequence analysis. RESULTS: PCR analysis, enzyme digestion and DNA sequence analysis proved the pAdTrackU6shRNA and the pAdU6shRNA had been successfully constructed. After being packaged in 293 cells, the recombinant adenovirus AdenoU6shRNA was identified by PCR analysis and DNA sequence analysis. CONCLUSION: We have successfully constructed recombinant adenovirus AdenoU6shRNA targeted against the rat Nogo66 receptor gene. It will be helpful to use RNAi in the research of the role of NgR in neural regeneration after cerebral ischemic injury.
【Keywords】 Nogo66 receptor; RNAi; adenoviruses
0引言
缺血性脑损伤后中枢神经的再生是神经康复难以逾越的障碍，近来研究发现，中枢神经再生困难的原因是CNS三种轴突再生抑制蛋白的存在，它们是NogoA,MAG和OMgp，与共同的受体NgR结合，激活下游信号及GTPase Rho系统，导致轴突生长锥崩溃［1］. 因此，NgR成为理想的治疗靶. RNA干扰(RNAi)是由双链RNA介导的、在转录后mRNA水平关闭相应基因表达过程-将与靶基因同源的21~23 bp的双链RNA导入细胞,它在细胞中可与靶基因mRNA结合,并迅速将其降解,从而抑制该基因的表达的过程［2］.而成功地进行RNAi的关键在于使siRNA导入细胞，我们利用前期实验已构建的NgR shRNA真核表达载体，进一步构建腺病毒表达载体，通过病毒载体导入NgR siRNA沉默NgR基因，从而达到拮抗NgR基因的效应，促进神经再生.
