Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor

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Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor

更新时间：2026-01-09

- Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor
- Journal of Army Medical University Vol. 29, Issue 13, Pages: 1285-1288(2007)
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  CLC： R392-33;R392.11
- Published：2007
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范有明,蔡明春.Nogo-66受体RNA干扰质粒的构建及其干扰效率鉴定[J].陆军军医大学学报(原第三军医大学学报),2007,29(13):1285-1288.

　FAN You-ming,CAI Ming-chun.Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(13):1285-1288.
范有明,蔡明春.Nogo-66受体RNA干扰质粒的构建及其干扰效率鉴定[J].陆军军医大学学报(原第三军医大学学报),2007,29(13):1285-1288. DOI：

　FAN You-ming,CAI Ming-chun.Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(13):1285-1288. DOI：

摘要

目的构建及筛选高效率针对Nogo-66受体（Nogo-66 receptor

NgR）进行RNA干扰（RNAi）的质粒。方法 RT-PCR克隆NgR，插入表达质粒中，构建NgR-GFP融合蛋白表达质粒。根据NgR序列，设计4对针对NgR mRNA进行RNAi的寡核苷酸。退火后，插入短发夹RNA（short hairpin RNA

shRNA）表达质粒中，构建shRNA表达质粒。将NgR-GFP融合蛋白表达质粒与shRNA表达质粒共转染AAV-293细胞。通过对GFP表达量的观察及Western blotting定量检测NgR-GFP融合蛋白表达量，鉴定shRNA表达质粒对NgR的干扰效率。结果针对NgR进行RNAi的4个序列中，有1个序列的干扰效率大于90%以上。结论成功构建了1个针对NgR的高效RNAi表达质粒。

Abstract

Objective To construct and identify of a plasmid mediated high efficiency of RNA interference (RNAi) against Nogo-66 receptor (NgR). Methods After cloning of NgR by RT-PCR

the fragments were inserted into pcDNA3.1/CT-GFP-TOPO to produce plasmid expressing NgR-GFP fusion protein. Four pairs of oligonucleotide were designed according to NgR sequence and annealed. The resulting fragments were ligated into short hairpin RNA (shRNA) expressing plasmid. The plasmids expressing NgR-GFP fusion protein and shRNA were co-transfected into AAV-293 cells. RNAi efficiency against NgR was observed under fluorescent microscope and calculated by Western blotting. Results The interference efficiency of one sequence was above 90%. Conclusion A plasmid mediated high RNAi efficiency against NgR is constructed successfully.

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