Nogo-66拮抗性多肽的筛选及其对脊髓损伤大鼠轴突再生的影响

Selection of peptide binding specifically to the Nogo-66 by ribosome display and its infuence on axonal regeneration of injured spinal cord

目的:筛选与Nogo-66特异性结合的多肽,并进行初步功能检测。方法:采用了核糖体展示技术,即体外人工合成引物和含36个随机序列的寡核苷酸,通过两轮PCR构建随机DNA文库,然后在体外进行偶联的转录和翻译,得到含随机12肽的核糖体文库,并利用含随机12肽的核糖体文库对Nogo-66进行初步筛选。用ELISA方法检测筛选到的多肽与Nogo-66的亲和力。同时制备大鼠T10节段挫伤模型,损伤后1d尾静脉注射筛选到的多肽(50g/50l),HRP逆行染色观察脊髓轴突再生情况,并通过BBB评分观察大鼠神经功能恢复。结果:经过10轮筛选,得到了可与Nogo-66结合的氨基酸序列,合成相应的多肽NAPA,同时合成对照多肽NAPB。ELISA结果显示,NAPA与Nogo-66的亲和力明显高于NAPB与Nogo-66的亲和力(P(0.05)。NAPA尾静脉注射4、6周时,实验组大鼠BBB评分明显高于对照组(P(0.05),HRP逆行示踪显示NAPA治疗组的脊髓髓鞘染色清晰,排列规则。结论:筛选得到的Nogo-66的配体多肽NAPA可以明显促进脊髓损伤动物的恢复及脊髓再生。多肽NAPA的筛选成功为脊髓损伤的修复提供新的治疗方法和药物选择。

Objective：To select the peptide that bind specifically to Nogo-66 by ribosome display and examine its effect to the regeneration of spinal cord.Methods：36 random-sequence oligonucleotides and primers were designed and synthesized.A DNA random library was constructed by PCR using a random-sequence oligonucleotides template.Then a coupled transcription/translation reaction in vitro was proceeded by the use of E.coli S30 Extract System.The ribosome display system including 12 random peptides was used to screen the inhibitory peptide of Nogo-66.The affinity between Nogo-66 and the selected peptide was determined by ELISA methods.Spinal injury at T10 level was established and selected peptides（50 g/50 l）were injected through tail vein 1 d post-injury.HRP tracing method was used to observe the regeneration of the axons and the recovery of nervous function was examined by BBB scoring.Results：after ten rounds selection,the sequence of the peptide binding specifically to the nogo-66 protein was obtained and corresponding peptides,NAPA and NAPB,were synthesized.ELISA results showed that the affinity between NAPA and Nogo-66 was higher than that of NAPB（P 0.05）.Following injection by tail vein,BBB scoring of rats in the experiment group was higher significantly than that of the control group at 4,6 weeks post-injury.HRP staining showed better axon regeneration in the experiment group.Conclusion：the peptide NAPA can selectively bind to Nogo-66.The NAPA can prompt the injuried axons to regenerate and thus to improve the recovery of spinal cord injury,which will provide a new method for the therapy of spinal cord injury.