CN105061560A - Nogo-A receptor binding peptide as well as derivative and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a Nogo-A receptor binding peptide as well as a derivative and an application thereof. The amino acid residue sequence of the Nogo-A receptor binding peptide is HIYTALV or GFATITG. The derivative of the binding peptide is a product obtained through conventional modification of an amino terminal or a carboxyl terminal of a Nogo-A receptor binding peptide fragment on a Nogo-A receptor binding peptide amino acid side chain group or a product obtained by connecting a label used for peptide or protein detection or purification on the Nogo-A receptor binding peptide. The binding peptide and the derivative of the binding peptide can be bound with NgR in vitro, nerve regeneration is promoted by preventing binding of NgR and Nogo-A, nerve regeneration barriers caused by central nervous injury diseases can be treated, recovery of a central nervous function is promoted, and the binding peptide and the derivative can be widely applied in the medical and biological fields.

Description

A kind of Nogo-A receptor-binding peptides and derivative thereof and application

Technical field

The invention belongs to biomedicine field, be specifically related to a kind of Nogo-A receptor-binding peptides and derivative thereof and application.

Background technology

Along with the prolongation of human longevity, the problem of an aging population becomes increasingly conspicuous, and the disease incidence of some central nervous systems (centralneveroussystem, CNS) is more and more higher, and effectively treatment means lacks.The human body headquarters of major cause to be central nervous system be process information, its function depends on the neural network that a large amount of fundamental structural unit neurones is formed.Because neurone itself can not divide, once neuronal damage can only reach reconnecting of neural network by the regrowth of nervous process, and then reach the recovery of function, and the ability of injured neurons regeneration itself is very weak, which results in main central nervous system injury disease does not have good curative effect.Central nervous system injury disease includes neurotrauma, cerebral apoplexy, nerve degenerative diseases etc.Current this kind of disease does not also have effective medicine that the neural function of patient can be allowed to be recovered.Have very strong regenerative power after mammal peripheral nerve (peripheralneveroussystem, PNS) damage, and central nervous system (CNS) regenerative power is very poor, the projection after damage and neurone almost can not regenerate.For this reason, the disease such as CNS wound, apoplexy all can not recover its sensation, motion and cognitive function completely.As far back as 1913, Cajal just recognized that the ability of neurotization is subject to externalities restriction.If he observes damage tractus opticus, tractus opticus itself does not have regenerative power.If peripheral nerve graft is sewn onto injury region, some projections can grow into.He thinks, it is that neuroglia environment residing for neurone causes that Central Pathway can not regenerate.Many years go over, and the observation of Cajal is forgotten by people.Until 1970, many clinical and neurologists believe that central nerve regeneration is impossible.

1988, Schwab and Caroni is at cultivation Dorsal Root Ganglion Neurons (dorsalrootganglia, and add in differentiation-inducing neuroblastoma and derive from the sphingomyelinase of PNS or the sphingomyelinase of CNS or oligodendrocyte DRG), the sphingomyelinase of discovery CNS or oligodendrocyte can cause the collapse of growing tip, enation is suppressed, and the sphingomyelinase deriving from PNS does not affect its growth.This shows that the aregeneratory of CNS is relevant with the sphingomyelinase of CNS.They isolate size by SDS-PAGE method from adult central myelin is 35kD and 250kD two protein, is named as NI-35/250, has the effect suppressing DRG and neuroblastoma enation especially.And the regeneration of corticospinal pathway after Spinal injury is improved to a certain extent with this antibody capable prepared.

2000,3 research groups independently determined the encoding sequence of NI-250, and called after Nogo-A.Nogo gene can produce 3 kinds of varient: Nogo-A (1162 amino acid) by different montages, Nogo-B (373 amino acid) and Nogo-C (199 amino acid).Their C-end contains 188 identical amino acid, and these 188 amino acid and reticulon (RTN) gene family member have homology.The family that the protein that Reticulon is associated by endoplasmic reticulum forms, most of protein function is not clear, and in order to distinguish with other 3 kinds that find, Nogo albumen is otherwise known as RTN4.3 Nogo isomer contain two hydrophobic regions, make protein twice cross-film.Cell surface intermediate ring region between Liang Ge cross-film district, has a segment length to be 66 amino acid whose peptide sections, is called Nogo-66, and it has the activity suppressing axon growth.Another region with suppression enation activity of Nogo-A is Nogo-A _544-725.It is in the N-terminal of protein, so be also referred to as amino-Nogo-A (amino-Nogo-A).Soon just have found the acceptor (nogo-66receptor, NgR) of the Nogo-66 be combined with Nogo-66.Found again the NgR of two other homology afterwards, the acceptor be therefore combined with Nogo-66 was otherwise known as NgR1 afterwards.Nogo-A mainly expresses in oligodendrocyte, but also can express in many neurones.

Except Nogo-A, the growth-inhibiting molecule in another two myelins source, Myelin-associated glycoprotein (oligodendrocyte-myelinglycoprotein, and oligodendrocyte myelin glycoprotein (myelin-associatedglycoprotein OMG), MAG) also in conjunction with NgR, retarding effect can be produced.NgR is the protein of a kind of glycosyl-phosphatidyl inositol anchor fixed (glycosylphosphatidyl-anchored, GPI-anchored), is made up of 473 amino acid.This albumen n end is rich in the structural domain of leucine tumor-necrosis factor glycoproteins (leucine-richrepeat, LRR), and its C-terminal is rich in halfcystine structure.NgR is attached to surface of cell membrane by means of the GPI structure of C-terminal.NgR is not transmembrane protein, but the protein of GPI grappling, participate in intracellular delivery signal demand other oroteins, the common receptor complex that formed carrys out conducted signal.P75NTR and LINGO-1 in NgR receptor complex can suppress signal by transduce growth.Further research finds, at Tumor Necrosis Factor Receptors family member---the TAJ/TROY that many adult neuronal are expressed, can replace p75NTR, as the co-receptor of NgR1.

Closing Nogo-66 acceptor can make neurone insensitive and exempt from axon regeneration obstacle to Nogo-66.Thus, infer that blocking-up Nogo-66 and its receptors bind will contribute to the recovery of mankind's central nervous system injury.Nogo-66 acceptor also just becomes a potential target of disease such as treatment CNS trauma, apoplexy etc.Use Nogo-66 receptor antagonist NEP1-40, the suppression of Nogo-A to enation can be weakened.When after Spinal Cord Injury in Rats, give NEP1-40 treatment and can promote tractus corticospinalis and raphe-spinal tract regeneration, animal movement function is obviously improved.After mouse local cerebral infraction, use NEP1-40 can improve the regeneration of aixs cylinder, motor function recovery.Also observe with it at Nogo-66 acceptor gene knock-out mice, after Spinal injury, Monakow's bundle and raphe-spinal tract regeneration strengthen.

The effect of Nogo-A in central nervous system disease is also confirmed, suppresses the expression of Nogo-A to be conducive to axon growth and recovery in Spinal injury and apoplexy.IN-1 monoclonal antibody has the effect of anti-nogo-a, have simultaneously stronger in and axon growth restraining effect.There are some researches show and inject IN-1 after Spinal injury, nerve fiber regeneration distance increases and function obtains recovery, and IN-1 also can cause neuronic growth after apoplexy.More have for Nogo-A albumen Special Areas such as N-terminal and C-terminal targeting antibodies, after apoplexy can be conducive to function improvement and be conducive to tractus corticospinalis regeneration.The card base of a fruit and Ma Kusi etc. study discovery, in the mouse of apoplexy, Nogo-A or Nogo-66 acceptor (NgR) genetic flaw experimental group and the mouse taking NgR antibody and Nogo-A antagonist group have stronger neural compensatory and germinate and structure plasticity.

Nogo-A is activated by the interaction of multidigit point by specific binding acceptor NgR, and the signal transduction pathway started in neurocyte suppresses the remoldability of axon regeneration and structure, thus plays a role in hypoxic-ischemic brain injury disease develops.After there are some researches show rat cerebral ischemia anoxic and expression of receptor all increase, the regeneration of strongly inhibited nerve fiber, participates in cerebral ischemia a series of pathophysiological process.Lang Jia etc. study discovery, and global brain ischemia causes and significantly strengthens in the expression of cortex, and hyperbaric oxygentherapy significantly can alleviate nerve injury, reduction and the expression at cortex.The research such as Zhao Hong shows, early stage in cerebral infarction, expresses and strengthens, and application antibody obviously can promote impaired central nervous system axon growth and functional rehabilitation.These researchs show, regulation and control Nogo-A/NgR can affect development and the result of cerebral ischemia.

Research in recent years shows, Nogo-A and NgR has certain effect in (AD and PD) morbidity in neurological disease.The reports such as Gil and Zhu in 2006, in AD hippocampal tissue, Nogo-A is process LAN, and is associated with senile plaque, and Nogo-66 acceptor is also process LAN, is associated with neurofibrillary tangles.The same year, Park etc. was in succession at " TheJournalofNeuroscience " upper report, and in AD patient's brain, the distribution of Nogo-A and Nogo-66 acceptor all exception occurs.Find that NgR and APP interaction can reduce amyloid (amyloidbetaprotein, A β) and precipitate the senile plaque formed simultaneously.Show as and stop NgR to express the level then increasing APP in AD transgenic models mouse brain and the senile plaque increasing A β formation of deposits, make projection nutrition generation obstacle, then reduce A β level to these mouse perfusion solubility NgR segment and A β precipitates the senile plaque formed, the underfed situation of projection be improved significantly.Turning the SH-SY5Y clone of app gene, NgR overexpression can reduce A β too and produce.More than research prompting Nogo-66 acceptor can be used as the blocker in APP treating processes.Nogo-66 acceptor provides the new target drone that can be intervened AD pathologic process, makes people pay close attention to projection dysfunction role in this disease.

How research promotes neuronic regenerative power, and recovering neural network becomes the key that treatment nerve injury is treatment nerve injury.Blocking-up Nogo-66 and its receptors bind will contribute to the recovery after central nervous system injury.Therefore, Nogo-66 acceptor also just becomes a potential target of disease such as treatment CNS trauma, apoplexy etc.

Along with the quickening of Chinese society aging, incidence and the morbidity of neurotrauma, cerebral apoplexy and nerve retrograde affection raise year by year.These diseases bring huge financial loss to society, immeasurable burden is brought to sufferer individual and family, therefore research treatment nerve damage is sick sick, and the specifics as neurotrauma, cerebral apoplexy and nerve degenerative diseases has important social reality meaning.

Summary of the invention

In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the present invention is to provide a kind of Nogo-A receptor-binding peptides, this binding peptide and Nogo-A acceptor (NgR) have specificity high-affinity, and the binding site of Specific competition Nogo-A and Nogo-A acceptor, Nogo-A can be suppressed in conjunction with NgR.

Another object of the present invention is to the derivative that above-mentioned Nogo-A receptor-binding peptides is provided, this derivative also can have specificity high-affinity with Nogo-A acceptor, and the binding site of Specific competition Nogo-A and Nogo-A acceptor, Nogo-A can be suppressed in conjunction with NgR.

Another object of the present invention is the application providing above-mentioned Nogo-A receptor-binding peptides and derivative thereof.

Object of the present invention is achieved through the following technical solutions:

A kind of Nogo-A receptor-binding peptides, its amino acid residue sequence is:

His-Ile-Tyr-Thr-Ala-Leu-Val (HIYTALV) or Gly-Phe-Ala-Thr-Ile-Thr-Gly (GFATITG);

The derivative of described Nogo-A receptor-binding peptides is on Nogo-A receptor-binding peptides amino acid side groups, the aminoterminal of Nogo-A receptors bind peptide fragment or carboxyl terminal carry out conventionally modifying the product obtained, or the product obtained for label Nogo-A receptor-binding peptides connected for polypeptide or Protein Detection or purifying;

Described routine is modified and is preferably amination, amidation, hydroxylation, carboxylated, carbonylation, alkylation, acetylize, phosphorylation, esterification, glycosylation, cyclisation, biotinylation, fluorophor are modified, polyoxyethylene glycol PEG modifies or immobilization modification etc.;

Described label is preferably His ₆, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact etc.;

The derivative of described Nogo-A receptor-binding peptides is preferably:

Above-mentioned Nogo-A receptor-binding peptides second amino-acid residue is D configuration Isoleucine, and end carries out amidation modification, is His-(d) Ile-Tyr-Thr-Ala-Leu-Val-NH ₂;

Or above-mentioned Nogo-A receptor-binding peptides second amino-acid residue is D configuration phenylalanine, end carries out amidation modification, is Gly-(d) Phe-Ala-Thr-Ile-Thr-Gly-NH ₂;

Described Nogo-A receptor-binding peptides and the acquisition of derivative thereof, known method of the prior art is adopted to carry out, both can carry out chemosynthesis with polypeptide automatic DNA synthesizer DNA, and by short peptide sequence is derived nucleotide sequence, then be cloned in carrier and carry out biosynthesizing;

Described Nogo-A receptor-binding peptides and derivative thereof can be applied to the medicine that preparation prevents and/or treats central nervous system injury disease aspect;

Preferably, described Nogo-A receptor-binding peptides and derivative thereof can be applied to the medicine that preparation prevents and/or treats at least one in neurotrauma, cerebral apoplexy and nerve degenerative diseases three kinds of diseases;

Described nerve degenerative diseases is preferably alzheimer's disease or Parkinson's disease etc.;

Described medicine can containing a kind of or multiple pharmaceutically acceptable carrier;

Described carrier is preferably thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, absorption carrier, tensio-active agent or lubricant etc.;

Described medicine can make the various ways such as tablet, granula, capsule, oral liquid or injection further, and the medicine of various formulation can be prepared according to the ordinary method of pharmaceutical field;

Prevent and/or treat a medicine for central nervous system injury disease aspect, comprise at least one in the derivative of above-mentioned Nogo-A receptor-binding peptides and Nogo-A receptor-binding peptides;

The present invention has following advantage and effect relative to prior art:

(1) the invention provides a kind of Nogo-A receptor-binding peptides and derivative thereof, described Nogo-A receptor-binding peptides and derivative thereof can specificity and the single-minded combinations of NgR, and Specific competition Nogo-A and NgR binding site, Nogo-A can be suppressed in conjunction with NgR.

(2) the present invention screens the Nogo-A receptor-binding peptides that obtains and derivative in vitro can in conjunction with NgR, neurotization is promoted by blocking the combination of NgR and Nogo-A, the disease for the treatment of neurotization obstacle, promote the recovery of central functions, can as the biological species polypeptide drugs of NgR and Nogo-A binding site on neurocyte, can be used for preparing the medicine preventing and/or treating central nervous system injury disease aspect, such as: neurotrauma, cerebral apoplexy and nerve degenerative diseases.Can be widely used in Med Biol field, and produce huge social and economic effects.

Accompanying drawing explanation

Fig. 1 is the HPLC detection figure spectrogram of His-Ile-Tyr-Thr-Ala-Leu-Val.

Fig. 2 is the MS detection figure spectrogram of His-Ile-Tyr-Thr-Ala-Leu-Val.

Fig. 3 is the HPLC detection figure spectrogram of Gly-Phe-Ala-Thr-Ile-Thr-Gly.

Fig. 4 is the MS detection figure spectrogram of Gly-Phe-Ala-Thr-Ile-Thr-Gly.

Fig. 5 is the interpretation of result figure that ELISA method detects phage-displayed polypeptides and NgR specific binding.

Fig. 6 be Nogo-A receptor-binding peptides in different cell with the interpretation of result figure of NgR specific binding; Wherein, the interpretation of result of A:2 polypeptide (NAP2) and different Cell binding; The interpretation of result that B:2 polypeptide (NAP2) and PC-12 cell surface NgR locate altogether; The fluorescence semi-quantitative analysis of C:2 polypeptide (NAP2) and different Cell binding ability; D:2 polypeptide (NAP2) is total to the Fluorescence Intensity Assays of site binding ability with PC-12 cell.

Fig. 7 is the confocal detection interpretation of result figure of Nogo-A receptor-binding peptides and Nogo-66 competition binding NgR; Wherein, A ~ D: No. 2 polypeptide (NAP2) of different concns and Nogo-66 and the PC12 Cell binding ability (A:PBS of fixed concentration; B:1 μM of No. 2 polypeptide; C:4 μM of No. 2 polypeptide; D:16 μM of No. 2 polypeptide); The sxemiquantitative fluorometric analysis of the fluorescence intensity of E:2 polypeptide (NAP2) and Nogo-66.

Fig. 8 be Nogo-A receptor-binding peptides in PC-12 cell antagonism Nogo-66 to the interpretation of result figure of the provide protection that nervous process damages; Wherein, the provide protection (a:PBS that the nervous process that A:2 polypeptide (NAP2) causes Nogo-66 in PC-12 cell damages; B:0.3 μM of Nogo-66; C:0.3 μM of Nogo-66 and 1 μM of No. 2 polypeptide; D:0.3 μM of Nogo-66 and 4 μM of No. 2 polypeptide; E:0.3 μM of Nogo-66 and 16 μM of No. 2 polypeptide; F:0.3 μM of Nogo-66 and 1 μM NEP1-40); The cell viability of B:2 polypeptide (NAP2); C: the projection that the Nogo-66 of different concns causes shortens; The disappearance of the branch that D:2 polypeptide (NAP2) antagonism is caused by Nogo-66, promotes the increase of projection branch; E:2 polypeptide (NAP2) antagonism Nogo-66 causes the shortening of projection, promotes the growth of Neuronal processes, facilitates the regeneration of neurocyte.

Fig. 9 be Nogo-A receptor-binding peptides in CGCs cell antagonism Nogo-66 to the interpretation of result figure of the provide protection that nervous process damages; Wherein, the provide protection (a:PBS that the nervous process that A:2 polypeptide (NAP2) causes Nogo-66 in CGC cell damages; B:0.3 μM of Nogo-66; C:0.3 μM of Nogo-66 and 1 μM of No. 2 polypeptide; D:0.3 μM of Nogo-66 and 4 μM of No. 2 polypeptide; E:0.3 μM of Nogo-66 and 16 μM of No. 2 polypeptide; F:0.3 μM of Nogo-66 and 1 μM NEP1-40); B: the projection that the Nogo-66 of different concns causes shortens, and the disappearance of the branch that C:2 polypeptide (NAP2) antagonism is caused by Nogo-66, promotes the increase of projection branch; D:2 polypeptide (NAP2) antagonism Nogo-66 causes the shortening of projection, promotes the growth of Neuronal processes, facilitates the regeneration of neurocyte.

Figure 10 is the interpretation of result figure of Nogo-A receptor-binding peptides on the ROCK2 cell-signaling pathways impact that Nogo-66 activates; Wherein, A, B: the expression adding ROCK2 after No. 2 polypeptide (NAP2) of different concns and Nogo-66 and NEP1-40 and Nogo-66 in PC-12 and CGCs cell; C, D: the gray-scale value analysis adding ROCK2 after No. 2 polypeptide (NAP2) of different concns and Nogo-66 and NEP1-40 and Nogo-66 in PC-12 and CGC cell.

Figure 11 is Nogo-A receptor-binding peptides on the interpretation of result figure of the MLC cell-signaling pathways impact of the CRMP2 of the phosphorylation that Nogo-66 activates and phosphorylation; Wherein, E, F: the expression adding the CRMP2 of phosphorylation and the MLC of phosphorylation after No. 2 polypeptide (NAP2) of different concns and Nogo-66 and NEP1-40 and Nogo-66 in PC-12 and CGCs cell; G ~ J: the gray-scale value analysis adding the CRMP2 of phosphorylation and the MLC of phosphorylation after No. 2 polypeptide (NAP2) of different concns and Nogo-66 and NEP1-40 and Nogo-66 in PC12 and CGCs cell.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

The organic synthesis of embodiment 1Nogo-A receptor-binding peptides and detection

Organic synthesis No. 2 polypeptide (His-Ile-Tyr-Thr-Ala-Leu-Val) and No. 5 polypeptide (Gly-Phe-Ala-Thr-Ile-Thr-Gly) (being synthesized by Shanghai Qiang Yao biotech firm), take KP resin 1g and be placed in reactor, add DMF swelling 30min, filtering DMF, by Fmoc amino acid, HBTU, each 0.4mmol and NMM0.6mmol of HOBT is dissolved in appropriate DMF, added in reactor, in 40 DEG C of concussion 2h, filtering reaction solution, replaces washing resin each 2 times with methyl alcohol, DMF, washes once with anhydrous diethyl ether; With volume fraction 20% piperidines/DMF eluant solution Fmoc base; Reaction 10min, filtering reaction solution methyl alcohol, DMF replaces washing resin each 2 times; Carry out the coupling of next amino-acid residue, hold first amino acid to hold extension, until small peptide completes to N by C so successively.The small peptide of synthesis is through the desalination of G10 post and HPLC purifying, and lyophilize is preserved.

The purity of No. 2 polypeptide and No. 5 polypeptide all reaches more than 98%; Wherein Fig. 1 ~ 4 are respectively No. 2 polypeptide and No. 5 polypeptide HPLC detection collection of illustrative plates and MS and detect collection of illustrative plates.

Embodiment 2ELISA detects phage display: NgR specificity, specificity are in conjunction with positive monoclonal

Wrap by 96 hole enzyme plates (Corning, company) with the sNgR albumen (R & D company of the U.S.) by PBS weaker concn being 2.5 μ g/mL, every hole 150 μ L, simultaneously with not wrapping by the hole of sNgR in contrast.4 DEG C of slow incubator overnight, incline coating buffer, and the massfraction adding PBS dilution is 1%BSA confining liquid, closes 1h; Add the anti-mono-clonal 1 × 10 sieved of fourth round ¹⁰phage (NewEnglandBiolabs, Beverly, MA) the 100 μ L of pfu/mL, the slow shaking table of room temperature is in conjunction with 1h; Then be that 0.1%PBST washs 6 times with massfraction, add the anti-M13 antibody of HRP-(NEB company) 100 μ L (1:5000 dilution), hatch 40min for 37 DEG C, PBST adds display substrate TMB (green skies company) 50 μ L colour developing 10min, 1MH after washing 6 times ₂sO ₄color development stopping, microplate reader 450nm detects OD value.According to the result of ELISA, the mono-clonal sequence stronger to specificity carries out ssDNA extraction and order-checking, then Bioedit software and ProtParam program is adopted to carry out sequential analysis and specificity analysis, finally obtain showing that sequence is that His-Ile-Tyr-Thr-Ala-Leu-Val phage (12) and Gly-Phe-Ala-Thr-Ile-Thr-Gly phage (21) OD value are respectively 1.3512 and 1.2538, show that itself and NgR have higher specificity, specificity binding ability, the results are shown in Figure 5.

The combination of the burnt Nogo-A receptor-binding peptides of embodiment 3 copolymerization and NgR high expressing cell PC-12, CGCs surface of cell membrane NgR, and Nogo-A receptor-binding peptides and PC-12 cell surface NgR locate altogether

Focusing board inoculation PC-12, CGCs cell altogether, wherein PC12 cell is purchased from Chinese Academy of Sciences's cell bank (ATCCCRL-1721), CGCs cell takes from rear 6 ~ 8 days Sprague-Dawley (SD) suckling mouses (SPF level, experimental center, Guangdong Province (SCXK Guangdong 2013-0002)) of birth; Each ware 10000 cells, cultivate 24h, PBS washes three times, paraformaldehyde fixes 15min, PBS washes 3 times, and BSA closes 1.5h, then with No. 2 polypeptide or No. 5 polypeptide (wherein the FITC mark of No. 2 polypeptide or No. 5 polypeptide is by the preparation of Shanghai Qiang Yao biotech firm) the incubated at room 2h (positioning experiment needs to add (1:250) NgR antibody altogether) of the FITC of 1 μM mark, PBST washes three times, and DAPI core contaminates.MDA-MB-231 cell (purchased from Chinese Academy of Sciences's cell bank, ATCCHTB-26) and PBS are as negative control, and result is as Fig. 6.

Result as shown in Figure 6A, add same concentrations No. 2 polypeptide (NAP2) in different cell (PC12 cell, CGCs cell, MDA-MB-231 cell), the PC12 cell of high expression level NgR and the fluorescence intensity of CGCs cell very high, and the MDA-MB-231 cell fluorescence intensity of low expression NgR is very weak, polypeptide A P7 (screen from other incoherent seven peptide storehouses, sequence is VPPLWPA) in contrast and PBS is also very low at the PC12 cell fluorescence intensity of high expression level NgR.As can be seen from Fig. 6 B, No. 2 polypeptide (NAP2) and surface of cell membrane NgR specific binding.Fig. 6 C is No. 2 polypeptide (NAP2) and the fluorescence semi-quantitative analysis (semi-quantitative analysis to the fluorescence of Fig. 6 A) of different Cell binding ability, Fig. 6 D is that No. 2 polypeptide (NAP2) are total to the Fluorescence Intensity Assays of site binding ability with PC-12 cell, and AP7 is as negative control.Above result shows, No. 2 polypeptide can with NgR specific binding.

Same experimental technique proves: the result of No. 5 polypeptide is similar to No. 2 polypeptide, can with NgR specific binding.

Embodiment 4Nogo-A receptor-binding peptides and Nogo-66 competition binding NgR

Focusing board inoculation PC-12 cell altogether, each ware 10000 cells, cultivate 24h, PBS washes three times, paraformaldehyde fixes 15min, PBS washes 3 times, BSA closes 1.5h, then with different concns FITC mark No. 2 polypeptide or No. 5 polypeptide (PBS, 1 μM, 4 μMs, 16 μMs) incubated at room 1h, add Nogo-66 (the RecombinantNogo-66viasolubleexpressionwithSUMOfusioninEs cherichiacoliinhibitsneuriteoutgrowthinvitro.ApplMicrobi olBiotechnol.2015Jul of 1 μM of Rho-123 mark, 99 (14): 5997-6007, then through the mark preparation of Shanghai Qiang Yao biotech firm) 30min, PBST wash three times, DAPI core contaminates, and the results are shown in Figure 7.

As can be seen from Fig. 7 A ~ D, along with the concentration of No. 2 polypeptide of FITC mark increases, green fluorescence is grow gradually, and the red fluorescence of the Nogo-66 of Rho-123 mark dies down gradually.Fig. 7 E is the fluorescence intensity of semi-quantitative analysis Fig. 7 A ~ D.Above result shows, No. 2 polypeptide can with Nogo-66 competition binding NgR.

Same experimental technique proves: the result of No. 5 polypeptide is similar to No. 2 polypeptide, can with Nogo-66 competition binding NgR.

Embodiment 5Nogo-A receptor-binding peptides is on the impact of nervous process aregeneratory model

Focusing board inoculation PC-12, CGCs cell altogether, each ware 10000 cells, cultivate 24h.Then PC-12 cell adds the NGF (humanrecombinantNGF, Sigma) of 100ng/mL.

The Nogo-66 of No. 2 polypeptide (NAP2) of continuous gradient dilution or No. 5 polypeptide (PBS, 1 μM, 4 μMs, 16 μMs) and 0.3 μM is added in PC-12, CGCs cell, wherein, the cell of the Nogo-66 of 1 μM of NEP1-40 (Calbiochem (EMDMillipore)) and 0.3 μM is added as positive control; Continue to cultivate 24h, then fix with the paraformaldehyde that massfraction is 4%, add microtubulin-resisting β-III antibody (1:800; Sigma-Aldrich), projection length Image-ProPlus computed in software, tests small peptide to the impact of PC-12 cell survival rate with MTT simultaneously.The results are shown in Figure 8, Fig. 9.

First detect the toxicity of No. 2 polypeptide (NAP2) in PC12 cell with mtt assay, find in requirement of experiment concentration, there is no toxicity (see Fig. 8 B).Then in PC12 cell, add different concns Nogo-66, result shows the restraining effect that 0.3 μM of Nogo-66 has 50%, and this concentration is exactly modeling concentration (see Fig. 8 C).When PC12 cell adds No. 2 polypeptide (NAP2) of different concns and the Nogo-66 of fixed concentration (0.3 μM) simultaneously, result display can the projection that causes of antagonism Nogo-66 be shortened in various degree with the increase of No. 2 polypeptide (NAP2) concentration, increases PC12 cell process length and PC12 cell arm number.No. 2 polypeptide (NAP2) have the function (see Fig. 8 A, Fig. 8 D and Fig. 8 E) similar to positive drug NEP1-40.

Fig. 9 be No. 2 polypeptide (NAP2) in CGCs cell antagonism Nogo-66 to the interpretation of result figure of the provide protection that nervous process damages.In CGCs cell, add different concns Nogo-66, the Nogo-66 that result shows 0.3 μM has good restraining effect, and this concentration is exactly modeling concentration (see Fig. 9 B).When PC12 cell adds No. 2 polypeptide (NAP2) of different concns and the Nogo-66 of fixed concentration simultaneously, result shows No. 2 polypeptide and can the projection that causes of antagonism Nogo-66 shorten, increase CGCs cell process length and CGCs cell arm number, wherein obviously can see that No. 2 polypeptide (NAP2) have the function similar to positive drug NEP1-40.(see Fig. 9 A, Fig. 9 C and Fig. 9 D).

Above result can find out that No. 2 polypeptide (NAP2) can the projection that causes of antagonism Nogo-66 can be shortened in PC12 and CGC cell, promote the projection length of neurocyte and the increase of branches, facilitate the regeneration of neurocyte, and there is obvious dose-dependently.Equally, experimental result shows, No. 5 polypeptide can the projection that causes of antagonism Nogo-66 can shorten in PC12 and CGCs cell, promote the projection length of neurocyte and the increase of branches, facilitate the regeneration of neurocyte, and there is obvious dose-dependently.

The result that embodiment 6Nogo-A receptor-binding peptides affects cell-signaling pathways

At 6 orifice plate inoculation PC-12 and CGCs cells, each plate inoculation 3.5 × 10 ⁶individual cell, cultivates 24h.Add the Nogo-66 effect 15min of No. 2 polypeptide (NAP2) of continuous gradient dilution or No. 5 polypeptide (PBS, 1 μM, 4 μMs, 16 μMs) and 0.3 μM, wherein, the cell of the Nogo-66 of 1 μM of NEP1-40 and 0.3 μM is added as positive control.Sucking-off substratum, then the PBS adding precooling washs three times, add cell pyrolysis liquid (RIPA:PMSF=100:1) again, hatch 40min for 4 DEG C, then collect albumen and be placed in 4 DEG C of centrifugal 5min of refrigerated centrifuge (12000g/min), add 5 × Buffer, make protein sample.Protein sample is being carried out electrophoresis, transferring film, the pvdf membrane obtained containing albumen closes 1h by the TBST room temperature containing massfraction being the skim-milk of 5%, then anti-ROCK2 antibody, anti-phosphorylation CRMP2 antibody and anti-phosphorylation MLC antibody is diluted (all purchased from CellSignalingTechnology with the TBST containing massfraction being 5%BSA, Inc. (Shanghai, China)), after adding above-mentioned primary antibodie, 4 DEG C of overnight incubation.Next day, after adding two anti-(1:2000) of goat-anti rabbit, with the colour developing of ECL test kit, by Quantity-One software analysis experimental result, result as shown in FIG. 10 and 11.

No. 2 polypeptide (NAP2) or No. 5 polypeptide significantly can reduce the expression of ROCK2, wherein, Figure 10 A ~ B is the expression of No. 2 polypeptide (NAP2) ROCK2 afterwards simultaneously adding Nogo-66 and different concns in PC-12 and CGCs cell, and Figure 10 C ~ D is the gray-scale value analysis of ROCK2 on PC-12 and CGC cell.No. 2 polypeptide (NAP2) or No. 5 polypeptide can reduce the expression level of the CRMP2 of phosphorylation and the MLC of phosphorylation, and have obvious dose-dependently.Wherein, Figure 11 E ~ F is after No. 2 polypeptide (NAP2) and Nogo-66 and NEP1-40 and Nogo-66 adding different concns in PC-12 and CGCs cell, the expression of the CRMP2 of phosphorylation and the MLC of phosphorylation.Figure 11 G ~ J is the gray-scale value analysis of the CRMP2 of phosphorylation and the MLC of phosphorylation in PC12 and CGC cell.Above result shows to further illustrate the cell-signaling pathways that No. 2 polypeptide (NAP2) or No. 5 polypeptide energy antagonism Nogo-66 activate the suppression projection regeneration that it has antagonism Nogo-66, facilitate the regeneration of neurocyte.

Above embodiment proves, No. 2 polypeptide (NAP2) or No. 5 polypeptide can the projection that causes of antagonism Nogo-66 shorten, and facilitate the regeneration of neurocyte, its mechanism is the downstream signaling molecule that polypeptide inhibits Nogo-66 to activate.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (10)

1. a Nogo-A receptor-binding peptides, is characterized in that: the amino acid residue sequence of described Nogo-A receptor-binding peptides is:

His-Ile-Tyr-Thr-Ala-Leu-Val or Gly-Phe-Ala-Thr-Ile-Thr-Gly.

2. the derivative of Nogo-A receptor-binding peptides according to claim 1, is characterized in that:

The derivative of described Nogo-A receptor-binding peptides is on Nogo-A receptor-binding peptides amino acid side groups according to claim 1, the aminoterminal of Nogo-A receptors bind peptide fragment according to claim 1 or carboxyl terminal carry out conventionally modifying the product obtained, or the product obtained for label Nogo-A receptor-binding peptides according to claim 1 connected for polypeptide or Protein Detection or purifying.

3. the derivative of Nogo-A receptor-binding peptides according to claim 2, is characterized in that:

Described routine is modified to amination, amidation, hydroxylation, carboxylated, carbonylation, alkylation, acetylize, phosphorylation, esterification, glycosylation, cyclisation, biotinylation, fluorophor is modified, polyoxyethylene glycol PEG modifies or immobilization is modified;

Described label is His ₆, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.

4. the derivative of Nogo-A receptor-binding peptides according to claim 2, is characterized in that:

The derivative of described Nogo-A receptor-binding peptides is: His-(d) Ile-Tyr-Thr-Ala-Leu-Val-NH ₂or Gly-(d) Phe-Ala-Thr-Ile-Thr-Gly-NH ₂.

5. Nogo-A receptor-binding peptides according to claim 1 is preparing the application in the medicine prevented and/or treated in central nervous system injury disease.

6. Nogo-A receptor-binding peptides according to claim 5 is preparing the application in the medicine prevented and/or treated in central nervous system injury disease, it is characterized in that:

Described medicine contains a kind of or multiple pharmaceutically acceptable carrier.

7. Nogo-A receptor-binding peptides according to claim 5 is preparing the application in the medicine prevented and/or treated in central nervous system injury disease, it is characterized in that:

Described medicine makes tablet, granula, capsule, oral liquid or injection further.

8. the derivative of the Nogo-A receptor-binding peptides described in any one of claim 2 ~ 4 is preventing and/or treating the application in the medicine in central nervous system injury disease.

9. the derivative of Nogo-A receptor-binding peptides according to claim 8 is preventing and/or treating the application in the medicine in central nervous system injury disease, it is characterized in that:

Described medicine contains a kind of or multiple pharmaceutically acceptable carrier.

10. the derivative of Nogo-A receptor-binding peptides according to claim 8 is preventing and/or treating the application in the medicine in central nervous system injury disease, it is characterized in that:

Described medicine makes tablet, granula, capsule, oral liquid or injection further.