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1、2010,26(1):88~92神经解剖学杂志ChineseJournalofNeuroanatomyNogo-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1*121111陈金荣,石增立,杨成,刘凤,石磊,刘巍,袁文丹(滨州医学院:1.病理生理学教研室,2.解剖学教研室,滨州256603)摘要目的: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
2、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
3、),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。关键词Nogo-66;核糖体展示技术;亲和筛选;轴突再生SelectionofpeptidebindingspecificallytotheNogo-66byribosomedisplayanditsinfuenceonaxonalregenerationofinjuredspinalcord1121111ChenJinrong,S
4、hiZengli,YangCheng,LiuFeng,ShiLei,LiuWei,YuanWendan(1.DepartmentofPathophysilolgy,2.DepartmentofAnatomy,BinzhouMedicalCollege,Binzhou,256603,China)AbstractObjective:ToselectthepeptidethatbindspecificallytoNogo-66byribosomedisplayandexamineitseffecttotheregenerationofspinalcord.M
5、ethods:36random-sequenceoligonucleotidesandprimersweredesignedandsynthe-sized.ADNArandomlibrarywasconstructedbyPCRusingarandom-sequenceoligonucleotidestemplate.Thenacou-pledtranscription/translationreactioninvitrowasproceededbytheuseofE.coliS30ExtractSystem.Theribosomedis-playsyst
6、emincluding12randompeptideswasusedtoscreentheinhibitorypeptideofNogo-66.TheaffinitybetweenNo-go-66andtheselectedpeptidewasdeterminedbyELISAmethods.SpinalinjuryatT10levelwasestablishedandselectedpeptides(50g/50l)wereinjectedthroughtailvein1dpost-injury.HRPtracingmethodwasusedtoobse
7、rvetheregen-erationoftheaxonsandtherecoveryofnervousfunctionwasexaminedbyBBBscoring.Results:aftertenroundsselec-tion,thesequenceofthepeptidebindingspecificallytothenogo-66proteinwasobtainedandcorrespondingpeptides,NAPAandNAPB,weresynthesized.ELISAresultsshowedthattheaffinitybetwee
8、nNAPAandNogo-66washigherthanthato
