CN101511873A - Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it - Google Patents

Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it Download PDF

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CN101511873A
CN101511873A CNA2007800320987A CN200780032098A CN101511873A CN 101511873 A CN101511873 A CN 101511873A CN A2007800320987 A CNA2007800320987 A CN A2007800320987A CN 200780032098 A CN200780032098 A CN 200780032098A CN 101511873 A CN101511873 A CN 101511873A
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fusion polypeptide
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李承揆
李尚揆
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ForHumanTech Co Ltd
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Abstract

Disclosed herein is a method of enhancing the efficiency of delivery into cells and local sites by inhibiting the secretion of neurotransmitters using a conjugate of a protein transduction domain (PTD) with domains binding to three kinds of proteins, which form a SNARE complex.

Description

The method that is used to suppress the fusion polypeptide of neurotransmitter secretion and sends this fusion polypeptide
Technical field
The present invention relates to by use by nexin transduction domain (PTD) with can be attached to the conjugate that the structural domain on three kinds of protein that can form the SNARE complex body forms and suppress the secretion of neurotransmitter, thereby promote it to be delivered in the cell efficient with local location.
Background technology
Secretion about neurotransmitter, by SSV albumen (synaptobrevin) (vesica related membrane protein, VAMP), these three kinds of protein of SNAP25 (the anti-people's cynapse of rabbit related membrane protein 25) and syntaxin (syntaxin) form SNARE complex bodys, and these three kinds of protein pass through Ca 2+Stimulation to cell exocrine; Described SSV albumen is present in the surface of the synaptic vesicle that carries neurotransmitter, and SNAP25 and syntaxin are present in (referring to Fig. 1) in the presynaptic lipid film.
Botulic neurotoxin is through being often used as the excretory inhibitor of neurotransmitter, it comprises several hypotypes, as botulic neurotoxin A, B, C, D, E and F, each hypotype is cut VAMP-2, SNAP25 and syntaxin at specific site, to disturb the formation of SNARE complex body, thereby suppress the secretion (Schiavo et al.Nature 359,832-835,1992) of neurotransmitter.
Because this botulic neurotoxin has very high toxicity, therefore, existing report, research concentrates on the peptide fragment that use is cut by above-mentioned botulic neurotoxin, to suppress the secretion of neurotransmitter.Yet, owing to only use a complete proteinic part, therefore, to compare with botulic neurotoxin, the segmental specificity of described peptide is lower usually, thereby has lower activity.In addition, compare with the speed that botulic neurotoxin is delivered in the cell, if the fragment of described peptide is bigger, then this peptide is delivered to the speed reduction in the cell, therefore, the efficient that it conducts in cell is lower, because it is (AntonioV.Ferror-Monitiel et al.FEBS letters, 435 of carrying out with the state that is attached to acceptor that botulic neurotoxin is delivered in the cell, 84-88,1998).
Need a kind ofly can both to send bioactive macromolecule effectively and do not damage the usual way (LA.Sternson, Ann.N.Y.Acad.Sci., 57,19-21 (1987)) of cell with external in vivo.The example of this method comprises: method (the P.Hoffmann et al.Immunobiol. that is used for the chemical addition of lipid peptide, 177,158-170 (1998)) or use alkaline polymer (as, polylysine and poly arginine) method (W-C.Chen et al., Proc.Natl.Acad.Sci., USA, 75,1872-1876 (1978)), but these methods also are not verified.In addition, it is reported, be transferred to (C.P.Leamon and Low in the cell with the form of folic acid-conjugate as the folic acid of translocator, Proc.Natl.Acd.Sci., USA, 88,5572-5576 (1991)), but folic acid is delivered in the tenuigenin also and does not confirm.In addition, Pseudomonas exotoxin (pseudomonas exotoxin) also is used as a kind of translocator (T.I.Prior et al., Cell, 64,1017-1023 (1991)).Yet these methods are not also confirmed clearly for biologically active substance to be sent effect and their the general suitabilities in intracellular conduction.Therefore, continue needing can be with biologically active substance in safer and more effective mode, is delivered in the tenuigenin of viable cell or the effective ways in the nucleus.
Result as to the research of this needs has proposed nexin transduction domain (PTDs).In the middle of them, as human immunodeficiency virus-1's (HIV-1) transcription factor transcribe trans-activating factor (Tat) albumen, obtained research the most completely.It is found that, when tat protein is made up of the individual amino acid of 47-57 (YGRKKRRQRRR) (having concentrated positive charge amino acid), 86 amino acid whose protein forms than total length can more effectively pass cytolemma (Fawell S.et al., Proc.Natl.Acad.Sci.USA 91:664-668 (1994)).Other example of PTDs comprises: by 267 of herpes simplex virus type 1 protein (HSV-1) Th-300 ThThe peptide that amino acid is formed (Elliott G.et al., Cell 88:223-233 (1997)), with feeler foot (ANTP) proteic the 339th to the 355th amino acid institute component peptide (Schwarze S.R.et al., Trends Pharmacol Sci.21:45-48 (2000)) by fruit bat.In addition, also find the artificial peptide formed by positive charge amino acid also effectively (Laus R.et al., Nature Biotechnol.18:1269-1272 (2000)).
Summary of the invention
Therefore, inventor of the present invention attempt using can with three kinds of albumen structure combining territories that can form the SNARE complex body, promote the efficient of in cell, sending, can interfering the combination between the described albumen fully, thereby suppress the secretion of neurotransmitter.In the present invention, for the efficient that promotes in cell, to send, by with a kind of nexin transduction domain with can be attached to three kinds of proteic structural domains that can form the SNARE complex body and combine, and prepare a kind of fusion polypeptide, and use the fusion polypeptide of this preparation, to suppress the secretion of neurotransmitter.
The aminoacid sequence that can form SSV albumen (VAMP-2), SNAP25 and the syntaxin of SNARE complex body is distinguished as follows:
SNAP25(SEQ?ID?NO:1):
MAEDADMRNE LEEMQRRADQ LADESLESTR RMLQLVEESK
DAGIRTLVML DEQGEQLERI
EEGMDQINKD MKEAEKNLTD LGKFCGLCVC PCNKLKSSDA
YKKAWGNNQD GVVASQPARV
VDEREQMAIS GGFIRRVTND ARENEMDENL EQVSGIIGNL
RHMALDMGN E IDTQNRQIDR
IMEKAQANKT RIDEANQRAT KMLGSG;
VAMP2(SEQ?ID?NO:2)
MSATAATAPP AAPAGEGGPP APPPNLTSV NR RLQQTQAQVD
EVVDIMRVNV DKVLERDQKL
SELDDRADAL QAGASQFETS AAKLKRKYWW KNLKMMIILG
VICAIILIII IVYFSS; With
Syntaxin (SEQ ID NO:3):
MKDRTQELRT AKDSDDDDDV TVTVDRDRFM DEFFEQVEEI
RGFIDKIAEN VEEVKRKHSA
ILASPNPDEK TKEELEELMS DIKKTANKVR SKLKSIEQSI
EQEEGLNRSS ALDRIRKTQH
STLSRKFVEV MSEYNATQSD YRERCKGRIO RQLEITGRTT
TSEELEDMLE SGNPAIFASG
IIMDSSISKQ ALSEIETR HS EIIKLENSIR ELHDMFMDMA
MLVESQGEMI DRIEYNVEHA
VDYVERAVSD TKKAVKYQSK ARRKKIMIII CCVILGIIIA
STIGGIFG
The 170th to the 206th amino-acid residue of SNAP25 (SEQ ID NO:1) (runic and have the part of underscore) is corresponding to the structural domain that can be attached to VAMP-2 (SEQ ID NO:4; SBD); The 29th to the 86th amino-acid residue of VAMP-2 (SEQ ID NO:2) (runic and have the part of underscore) is corresponding to the structural domain that can be attached to the SNAP25/ syntaxin (SEQ IDNO:5; VBD).In the present invention, use the inhibitor of the conjugate of the PTD with SBD (SEQ ID NO:4) or VBD (SEQ IDNO:5) as neurotransmitter secretion.
To achieve these goals, the invention provides a kind of fusion polypeptide, this fusion polypeptide comprises: have the cell-penetrating of height and the nexin transduction domain (PTD) that can send in skin, eyes etc. efficiently; With people source SNAP25 can be in conjunction with the structural domain of VAMP-2 or VAMP-2 can be in conjunction with the structural domain of syntaxin/SNAP25, promptly, be used to suppress the peptide that the SNARE complex body forms, in neurocyte, described SNARE complex body is formed to be used for the secretion of neurotransmitter.Fusion polypeptide of the present invention is characterised in that: this fusion polypeptide (for example can suppress neurotransmitter, catecholamine and vagusstoff) secretion, and the combination of PTD can significantly increase the efficient of neurotransmitter secretion and not change the cytotoxicity of described fusion polypeptide.
Fusion polypeptide of the present invention is meant the fusion polypeptide be made up of the PTD peptide of the peptide (being also referred to as " VBD " herein) of peptide that is connected with derived from human source SNAP-25 (being also referred to as " SBD " herein) or derived from human source VAMP-2 (be also referred to as " PTD-SBD or VBD ") herein.The structure of described fusion polypeptide can be represented by following formula 1:
[formula 1]
Figure A200780032098D00071
Wherein, [] PTDIt is self cell-penetrating (self-cell-penetrating) PTD peptide; R 1For being selected from least a amino acid whose side chain in the group of forming by following amino acid: tyrosine, L-Ala, arginine, Xie Ansuan, glycine and proline(Pro); N is the integer of 5-30; And the total amino acid above 30% is arginine residues.The example of PTD comprises: Hph-1 (YARVRRRGPRR (SEQ IDNO:6)), Sim-2 (AKAARQAAR (SEQ ID NO:7)), transcribe trans-activating factor (Tat) (YGRKKRRQRRR (SEQ ID NO:8)), Antp (RQIKIWFQNRRMKWKK (SEQID NO:9)), VP22 (DAATATRGRSAASRPTERPRAPARSAS RPRRPVE (SEQID NO:10)), R7 (RRRRRRR (SEQ ID NO:11)), MTS (AAVALLPAVLLALLAPAAADQNQLMP (SEQ ID NO:12)), pep-1 (KETWWETWWTEWSQPKKKRKV (SEQ ID NO:13)) etc.[] GlyConnect self cell-penetrating PTD peptide and SBD/VBD peptide, and can improve the flowability of described fusogenic peptide, PTD-SBD that penetrates with increase or the joint efficiency between VBD and the SNAPE complex body.M is the integer of 0-5.Although in formula 1, adopt glycine as joint, be understandable that and use various terminal to realize purpose of the present invention.
SBD (SEQ ID NO:4) and VBD (SEQ ID NO:5) are respectively the peptide that derives from people SNAP-25 and VAMP-2, and scope of the present invention comprises the fragment of these structural domains.What it will be appreciated by those skilled in the art that is to use wherein amino acid to be modified the sequence shown in SEQ ID NO:4 and 5 of (that is, amino acid is substituted, inserts or deletes), as the SBD and the VBD part of fusion polypeptide of the present invention.
According to the present invention, can be by between SBD or VBD peptide and PTD peptide, inserting about 3 glycine residues, be delivered to and be absorbed into efficient in the cell with promotion.
Simultaneously, the method that is used to prepare fusion polypeptide of the present invention can well known to a person skilled in the art that method carries out by use, for example adopts solid phase synthesis process or uses the synthetic method of recombinant expression system.Yet the method that is used to prepare fusion polypeptide of the present invention is not limited to the above-mentioned method of pointing out.
In first method, the synthetic of described fusion polypeptide can use the equipment that is used for organic synthesis to carry out.This method is the solid phase synthesis process (J.Am.Chem.Soc.85 of Merrifield, 2149-21,54 (1963))), in the method, use semi-automatic peptide synthesizer (Peti-Syzer Model PSS-510) to synthesize described fusion polypeptide by making the amino acid generation condensation that is positioned at C-end and N-end.
Described solid phase synthesis is that the C-terminal from polypeptide begins, by the amino protected amino acid of a α is coupled on the suitable resin.Herein, the amino protected amino acid of described α is coupled on hydroxymethyl resin or the chloromethyl resin by ester bond.Use the blocking group of Fmoc (9-fluorenylmethyloxycarbonyl), and the amino acid of Fmoc-protection can be commercially available from Beadtec Company as α amino.For example, arginic amino is activated.(the Fmoc-amino acid of the tertiary butyl (t-Bu) or Pmc (2,2,5,7,8-pentamethyl-dihydropyrane-6 (pentamethylchroman-6)) protection is as the active residue of glycine, Methionin or glutamine by suitable group in use.In this synthetic method of peptide, will be used to be connected to the aminoterminal blocking group of the peptide chain on the solid support resin, use reagent (for example, trichoroacetic acid(TCA) (TFA) or phenol) to remove subsequently.Described peptide can extract in TFA solution and is separated with diethyl ether, and can come purifying by high performance liquid chromatography (HPLC).
Second method is to use biological method to prepare the method for described fusion polypeptide.This method may further comprise the steps: (a) with the gene clone of described fusion polypeptide in the expression vector pPET that can express particular peptide, the recombinant vectors that can express described fusion polypeptide with preparation; Use described pPET-PTD-SBS (VBD) expression vector at a large amount of reorganization translocator-PTD-SBS (VBD) peptide of expression in escherichia coli, then the peptide of separation and purifying expression.In this, the encode base sequence that is used for preparing recombinant vectors of described fusion polypeptide can be easily derived from aminoacid sequence disclosed herein by those skilled in the art.In addition, be used for inserting the system of selection and the general technology detail file of the cloning vector of described base sequence, the recombinant expression vector that uses the clone technology preparation, the method for using recombinant vectors to transform described host cell to be transformed with the host cell of expressing the target fusion polypeptide, with recombinant vectors, in the method that transformed host cells is expressed the method for described target fusion polypeptide and collect described target fusion polypeptide from the product that obtains, be conspicuous for a person skilled in the art.
On the other hand, the invention provides the neurotransmitter inhibitor that comprises fusion polypeptide, described fusion polypeptide contains the people source SBD/VBD peptide protein that is connected with nexin transduction domain (PTD) peptide.
This neurotransmitter inhibitor can be applied to various fields, comprises the alleviating of pain, the minimizing of wrinkle, the minimizing at jaw angle etc.Especially, described neurotransmitter inhibitor can be delivered to partial position by PTD, for example, and skin.
Fusion polypeptide of the present invention can be delivered to efficiently in the cell and local location in, thereby suppress the secretion of neurotransmitter effectively.
Description of drawings
Fig. 1 represents the mechanism of neurotransmitter secretion.
Fig. 2 represents the result according to the HPLC analysis of synthetic polypeptide of the present invention.
Fig. 3 represents the cleavage point diagram of PTD conjugate.
Fig. 4 represents the agar gel photo of PTD-SBD and PTD-VBD.
Fig. 5 represents the cell-penetrating efficient of PTD conjugate.
Fig. 6 represents by being the mean value of 50 CMAPs that sciatic nerve write down of 2Hz repetitious stimulation with the frequency; The amount of the wave amplitude of CMAP (dY) expression Muscle contraction, the time course (tC) between the time point of time point that time representation stimulates and generation CMAP.
Fig. 7 represents the wave amplitude of CMAP.
Embodiment
The preparation of embodiment 1:PTD-SBD (VBD) conjugate
(1) preparation of PTD-SBD (VBD) conjugate
YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG (PTD-GGG-SBD polypeptide)
Use the fusion polypeptide (PTD-GGG-VBD polypeptide) of semiautomatic synthesizer (Peti-Syzer Model PSS-510) synthetic amino acid array according to the method for solid phase synthesis as YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL QAGASQFETSAAKLKR.In order to realize this purpose, the Rink Amide mbha resin of 0.1mmol is placed the reactor of a standard, in this reactor, add mole number and be the activatory Fmoc-G of 0.5mmol and Fmoc-R (Fmoc-G and Fmoc-R are the amino acid of first C-terminal of polypeptide to be synthesized), begin the synthetic of described polypeptide then.With amino acid whose order, with each corresponding amino acid condensation of 0.5mmol 3 times from C-terminal amino acid to N-terminal.The dimethyl formamide (DMF) that use contains 20% piperidines carries out three deprotection effects to Fmoc, and the time of deprotection is 10 minutes; With DMF washing 10 minutes, coupling 100 minutes.When coupling, use N-hydroxybenzotriazole (HOBT) and 1,3-DIC (DIC), and washed 10 minutes with DMF.
(2) separation of fusion polypeptide and purifying
After synthetic finishing, described polypeptide is placed the healthy and free from worry pipe (corning tube) of 50-mL, and be cooled to 0 ℃.Then, the mixture that will contain the TFA of the distilled water of thioanisole (thianisol), 0.5mL of the phenol of 0.75g, the 1 of 0.25ml (EDT), 0.25ml and 8.25ml joins in the described pipe, at room temperature reacts 4 hours.After reaction is finished, 30ml is lower than 50 ℃ ether joins by filtering in the solution after described resin separated from described peptide, precipitate described peptide, use twice of ether washing precipitation then.Precipitation drying with collecting is dissolved among the MeOH, and by the HPLC purifying.When purifying, use C18 analytical column 218TP54 (VyDac) and C18 preparative column 218TP1022 (VyDac).In addition, when purifying, use the condition of 1mL/min and 25mL/min, and use solvent orange 2 A (100%H 2O+0.01%TFA) and solvent B (the described peptide of wash-out of 100% acetonitrile+0.01%TFA).Acetonitrile is removed from described peptide, and described peptide is carried out lyophilize, thereby obtain highly purified peptide (referring to Fig. 2).
Embodiment 2: the expression vector that uses microbial expression systems produce and purifying fusion polypeptide
(1) preparation of expression vectors
In order to make up the base sequence of the described fusion polypeptide of coding, the Hind III cleavage site of N-end and the BamH I cleavage site of C-end are arranged among the pUC 19 (available from Invitrogen), thereby have made up a masterplate.For polyhistidyl label high expression level and that be easy to purifying that will make described fusion polypeptide is attached on the proteic base sequence of coding high expression level, use restriction enzyme HindIII to separate described masterplate, and use the Auiaquick purification kit to carry out purifying with BamH I.Use restriction enzyme HindIII and BamH I to handle expression vector pPET, and carry out purifying with the Auiaquick purification kit.Then the expression vector of purifying is cloned in the base sequence template of the described fusion polypeptide of coding of above-mentioned preparation, has prepared recombinant expression vector like this.In Fig. 3, provided the schematic gene mapping of described expression vector.
(2) expression of the preparation of intestinal bacteria transformant and fusion polypeptide and purifying
By the heat shock method for transformation and use the expression vector of above-mentioned preparation, (available from Invitrogen) transforms to e. coli bl21, then, the e. coli strains (1%) of the conversion of 1ml is inoculated in the LB substratum of 100mL, and under 37 ℃, shakes pre-cultivation 12 hours.Then, pre-culture is inoculated into 1, in the LB substratum of 000mL, cultivated 4 hours down at 37 ℃.With the IPTG of 1mM (isopropylthio-(isopropyl β-D-thiogalactopyranoside); GibcoBRL cat.#15529-019) joins in the substratum, to induce the expression of lactose operon, substratum was hatched 8 hours, to induce the expression of described fusion polypeptide.4 ℃ down and with 5,000rpm removes centrifugal 20 minutes of substratum then supernatant liquor and keeps precipitation.Then, described resolution of precipitate is being contained the isozyme of 1mg/mL (Sigma, (the 50mM NaH of damping fluid cat#L-7651) 2PO 4, 300mM NaCl, 10mM imidazole, pH8.0) in, left standstill on ice then 30 minutes.Then, use the hot processor for ultrasonic wave XL of system in intensity as under the 300W, handled described precipitation 10 seconds, cooled off then 10 seconds, repeat described ultrasonic and refrigerative process, reach 3 minutes up to ultrasonic total time.With 12, the centrifugal lysate of 000rpm 20 minutes is removed the destructive Bacillus coli cells under 4 ℃, and collects the lysate of purifying.The 50%Ni that in described lysate, adds 2.5mL 2+(Qiagen cat#30230), stirs mixture 1 hour with 200rpm under 4 ℃, so that described fusion polypeptide is attached to Ni-NTA agar syrup 2+On-NTA the agarose.Then, with mixture by 0.8 * 4cm chromatographic column (BioRad, cat#731-1550).Damping fluid 2 (50mM NaH with 4mL 2PO 4, 300mM NaCl, the imidazoles of 20mM pH8.0) washs the material twice that obtains, and uses damping fluid 3 (the 50mM NaH of 0.5mL then 2PO 4, 300mM NaCl, the imidazoles of 250mM, pH8.0) washing is four times, thereby collects described fusion polypeptide.The fusion polypeptide of collecting is carried out SDS-PAGE, and by examining the blue dyeing of Ma Shi, the result of analysis as shown in Figure 4 then.In Fig. 4, swimming lane 1 is the albumen of standard molecule weight; Swimming lane 2 is PTD-SBD; Swimming lane 3 is PTD-VBD.In described fusion rotein, add 2mL with ice-cooled damping fluid (4M NH 2OH, 0.4M K 2CO 3, 6M GuHCl pH9.0) makes and contains 2M NH in the damping fluid that obtains 2OH, 0.2M K 2CO 3, 3M GuHCl, pH9.0.Then, under 45 ℃ with 225rpm stirred solution 5 hours so that described fusion rotein is separated into fusion polypeptide and translocator, and by add 12N HCl with the pH regulator of described solution to 2-3, further stirred again 10 minutes.After reaction is finished, be 6.5 by adding 12.5N NaOH with the reaction soln pH that neutralizes, then by being filled with Ni 2+The post of-NTA agarose is to separate translocator from described solution.The solution that obtains is carried out gel-filtration, salt component is removed lyophilize then, thereby the fusion polypeptide of acquisition purifying from described solution.
Embodiment 3: fusion polypeptide suppresses the effect of neurotransmitter secretion in testing tube.
The fusion polypeptide that analysis prepares in embodiment 1 and 2 suppresses the effect of neurotransmitter secretion.For relatively more active, SBD and VBD two kinds of fragments have been prepared respectively.Described fragment sample is as follows:
Sample 1:SBD (SEQ ID NO:4)
Sample 2:VBD (SEQ ID NO:5)
Sample 3:Hph-1-GGG-SBD (SEQ ID NO:14)
Sample 4:Hph-1-GGG-VBD (SEQ ID NO:15)
Sample 5:Tat-GGG-SBD (SEQ ID NO:16)
Sample 6:Hph-1-GGG-SBDF1 (SEQ ID NO:17)
Sample 7:Hph-1-GGG-SBDF2 (SEQ ID NO:18)
Sample 8:Hph-1-GGG-VBDF1 (SEQ ID NO:19)
Sample 9:Hph-1-GGG-VBDF2 (SEQ ID NO:20)
Hph-1-GGG-SBD(SEQ?ID?NO:14)
YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG
Hph-1-GGG-VBD(SEQ?ID.NO.:15)
YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR
Tat-GGG-SBD(SEQ?ID?NO:16)
YGRKKRRQRRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG
Hph-1-GGG-SBDF1(SEQ?ID?NO:17)
YARVRRRGPRRGGGIMEKAQANKTRIDEANQRATKMLGSG
Hph-1-GGG-SBDF2(SEQ?ID?NO:18)
YARVRRRGPRRGGGKTRIDEANQRATKM
Hph-1-GGG-VBDF1(SEQ?ID?NO:19)
YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQK
Hph-1-GGG-VBDF2(SEQ?ID?NO:20)
YARVRRRGPRRGGGLSELDDRADALQAGASQFETSAAKLKR.
(contain the 2mM L-glutaminate at the F-12K of 15ml substratum (Gibco), 1.5g/L sodium bicarbonate, T75 (Nunc) solution of 15% horse serum and 2.5% foetal calf serum (FBS)) the PC12 cell (pheochromocytoma of thawing in, ATCC CRL-1721), and in this substratum, cultivated 2 days, so that cell density is 625,000 cell/cm 2Then, substratum is centrifugal, remove supernatant, the F-12K substratum of equal volume and each example pharmaceuticals are joined in the precipitation, mixture was hatched one day.Then, with 10 μ M Ca 2+Stimulate mixture, analyze the amount of excretory catecholamine by fluorescence HPLC.The concentration range of the example pharmaceuticals that adds is 10 -6-10 -5M.The result who detects is as shown in table 1 below.As can be seen from Table 1, the compound by formula 1 expression of the present invention has the effect of excellent inhibition neurotransmitter secretion.
[table 1] polypeptide is to the inhibition effect of neurotransmitter secretion
Figure A200780032098D00141
Negative control: do not stimulate
Positive control: use Ca 2+Stimulate but do not use medicine
Embodiment 4: the influence of sending in the conjugate pair cell
In order to analyze the influence of sending in the PTD conjugate pair cell, use fluorescent substance FITC (available from Sigma; Fluorescein 5-isothiocyanate; 27072-45-3) each SBDF1 peptide and unconjugated SBDF1 peptide that is combined with PTD is carried out mark, and (FACSCalibur, BectonDickinson) (referring to Fig. 5) analyzes by Facs.Its result is: the combination of PTD makes intracellular effect of sending increase about 50 times.
Embodiment 5:PTD conjugate is to the test of the effect of muscle paralysis
Because botulic neurotoxin and polypeptide of the present invention can cause the muscle paralysis by suppressing neurotransmitter, respectively botulic neurotoxin and described polypeptide are expelled in the epicnemial muscle of rat, and 1 week after injection, 2 weeks and 3 whens week, measure the wave amplitude (dY) of the CMAP (CMAP) of the epicnemial muscle that causes by electricity irritation.When measuring, in the right epicnemial muscle of big SD (available from Hyochang) rat of 8 weeks, inject botulic neurotoxin A (by the Allergan preparation, and available from Sigma), sample 1, sample 3 and the sample 6 of 5 μ l respectively.Specifically, the botulic neurotoxin of 400U is dissolved in the physiological saline of 1ml, with the solution of preparation 400U/ml, and the solution of injection 5 μ l in rat once.In addition, sample 1, sample 3 and sample 6 give rat with the amount of 10ng, 100ng and 500ng respectively.
In the measurement of CMAP, in the abdominal cavity of rat, inject ketamine/xylazine mixture, with anesthetized rat, then rat is placed on the table with prone position, keeping temperature is 30 ℃, and four limbs are fixed on this table.The hip joint of lower limb to be detected, knee joint and ankle joint respectively with 90 ° of angles fix; Recording electrode, a reference electrode and a ground-electrode are connected to epicnemial muscle, heel string and vola (foot sole) respectively.Thrust stimulating electrode (+) pin at the leg curved part, thrust stimulating electrode (-) pin at the pectineus of pubis part and near end of thighbone (proximal femur).The electric current that makes 1-5 μ A to stimulate sciatic nerve, causes the CMAP of epicnemial muscle by described stimulating electrode.With the frequency is 2Hz repetitious stimulation 50 times, induces CMAP, and its intensity is corresponding to 150% of the stimulus intensity that is shown as maximum amplitude.After having a rest 5 minutes, be 20Hz repetitious stimulation 50 times with the frequency, induce CMAP.Bioelectric amplifier and signal handling equipment (CyberAmp380, Axon Instruments, Inc.) in, these signals are amplified 50 times, and at 300-3000Hz place trap signal.Then, (Digidata1320, Axon Instruments Inc.) are input in the computer, and described signal is represented with the form of ripple by signal acquiring system with filtering signal.
Analysis is stored in the signal in the computer, and wave amplitude (dY) is for by with the frequency being the negative peak of average potential of the CMAPs that obtains of 2Hz repetitious stimulation 50 times and the value between the posivtive spike; Time is the time course (tC) (referring to Fig. 6) between the time point of the time point of electricity irritation and negative peak.
As described in Figure 7, CMAP shows that for the result of botulic neurotoxin, sample 1, sample 3 and sample 6 botulic neurotoxin of 5 units of injection can make muscle paralyse fully; When the administered dose of sample 1,3 and 6 increased, sample 1 was expressed more weak muscle paralysis effect, is about 20% of botulic neurotoxin effect, and sample 3 is then expressed the effect identical with botulic neurotoxin with sample 6.
Although described preferred embodiment of the present invention, but they only are used for illustrative purposes, what it will be appreciated by those skilled in the art that is, do not departing under the prerequisite of scope and spirit essence of the present invention disclosed in the accompanying claims, can carry out various changes, increase and replacement.
Industrial applicibility
As mentioned above, fused polypeptide of the present invention can be delivered in the cell and local location effectively, Thereby effectively suppress the secretion of neurotransmitter.
Sequence table
<110〉Forhuman Tech Co., Ltd.
<120〉method that is used to suppress the fusion polypeptide of neurotransmitter secretion and sends this fusion polypeptide
<130>IPN-34385
<160>20
<170>KopatentIn?1.71
<210>1
<211>206
<212>PRT
<213〉artificial sequence
<220>
<223>SNAP25
<400>1
Figure A200780032098D00171
Figure A200780032098D00181
<210>2
<211>117
<212>PRT
<213〉artificial sequence
<220>
<223>VAMP2
<400>2
Figure A200780032098D00182
<210>3
<211>288
<212>PRT
<213〉artificial sequence
<220>
<223〉syntaxin
<400>3
Figure A200780032098D00191
Figure A200780032098D00201
<210>4
<211>37
<212>PRT
<213〉artificial sequence
<220>
<223>SBD
<400>4
Figure A200780032098D00202
<210>5
<211>58
<212>PRT
<213〉artificial sequence
<220>
<223>VBD
<400>5
Figure A200780032098D00203
<210>6
<211>11
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1
<400>6
Figure A200780032098D00212
<210>7
<211>9
<212>PRT
<213〉artificial sequence
<220>
<223>Sim-2
<400>7
Figure A200780032098D00213
<210>8
<211>11
<212>PRT
<213〉artificial sequence
<220>
<223>Tat
<400>8
Figure A200780032098D00214
<210>9
<211>16
<212>PRT
<213〉artificial sequence
<220>
<223>Antp
<400>9
Figure A200780032098D00221
<210>10
<211>34
<212>PRT
<213〉artificial sequence
<220>
<223>VP22
<400>10
Figure A200780032098D00222
<210>11
<211>7
<212>PRT
<213〉artificial sequence
<220>
<223>R7
<400>11
Figure A200780032098D00223
<210>12
<211>26
<212>PRT
<213〉artificial sequence
<220>
<223>MTS
<400>12
<210>13
<211>21
<212>PRT
<213〉artificial sequence
<220>
<223>pep-1
<400>13
Figure A200780032098D00232
<210>14
<211>51
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-SBD
<400>14
Figure A200780032098D00233
<210>15
<211>72
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-VBD
<400>15
Figure A200780032098D00242
<210>16
<211>51
<212>PRT
<213〉artificial sequence
<220>
<223>Tat-GGG-SBD
<400>16
Figure A200780032098D00243
Figure A200780032098D00251
<210>17
<211>40
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-SBDF1
<400>17
<210>18
<211>28
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-SBDF2
<400>18
Figure A200780032098D00253
<210>19
<211>45
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-VDBF1
<400>19
Figure A200780032098D00261
<210>20
<211>41
<212>PRT
<213〉artificial sequence
<220>
<223>Hph-1-GGG-VBDF2
<400>20
Figure A200780032098D00262

Claims (5)

1, a kind of fusion polypeptide that is used for suppressing in vivo neurotransmitter secretion, this fusion polypeptide comprises: nexin transduction domain with can be attached to the structural domain (SEQ ID NO:4) of SNAP25 or can be attached to the conjugate of the structural domain (SEQ ID.NO:5) of VAMP-2; Described nexin transduction domain is made up of 5-30 amino acid, and arginic content is higher than 30%; The described structural domain that can be attached to SNAP25 all can form the SNARE complex body with the structural domain that can be attached to VAMP-2.
2, fusion polypeptide according to claim 1, wherein, described nexin transduction domain has any aminoacid sequence that is selected from the group of being made up of SEQ ID NO:6 to SEQ ID NO:12 and SEQ ID NO:13.
3, fusion polypeptide according to claim 1 and 2, wherein, described nexin transduction domain by joint with described can be in conjunction with the structural domain of SNAP25 or can be in conjunction with the structural domain combination of VAMP-2.
4, fusion polypeptide according to claim 3, wherein, described joint is made up of three glycine.
5, a kind of composition that is used to suppress neurotransmitter secretion, said composition contain claim 1 or 2 described fusion polypeptide as activeconstituents.
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CN103314012B (en) * 2010-08-20 2018-07-10 李尚揆 Fusion protein with transcriptional regulatory domain and protein transduction domain and the functional transcription factor inhibitor containing it
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