CN101511873B

CN101511873B - Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it

Info

Publication number: CN101511873B
Application number: CN2007800320987A
Authority: CN
Inventors: 李承揆; 李尚揆
Original assignee: ForHumanTech Co Ltd
Current assignee: ForHumanTech Co Ltd
Priority date: 2006-08-31
Filing date: 2007-08-24
Publication date: 2012-11-07
Anticipated expiration: 2027-08-24
Also published as: JP2010502592A; KR20090045269A; CN101511873A; KR101064915B1; WO2008026852A1

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

Be used to suppress the fusion polypeptide and the method for sending this fusion polypeptide of neurotransmitter secretion

Technical field

The present invention relates to through 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 and the efficient of 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; Said 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 often is used as the excretory suppressor factor of neurotransmitter; It comprises several kinds of hypotypes, and like 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 said peptide is lower usually, thereby has lower activity.In addition, compare, if the fragment of said peptide is bigger with the speed that botulic neurotoxin is delivered in the cell; Then this peptide is delivered to the speed reduction in the cell, and 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 (L.A.Sternson, Ann.N.Y.Acad.Sci., 57,19-21 (1987)) of cell with external in vivo.The instance of this method comprises: be used for the chemical addition of lipid peptide method (P.Hoffmann et al.Immunobiol., 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 are not also 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, PE (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 for biologically active substance to be sent effect and their the general suitabilities in intracellular conduction clearly.Therefore, continuation needs can be with biologically active substance with safer and more effective mode, is delivered in the tenuigenin of viable cell the effective ways in the perhaps 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.According to finding; 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 instance 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, the artificial peptide of also finding to be made up of positive charge amino acid is effectively (Laus R.et al., Nature Biotechnol.18:1269-1272 (2000)) also.

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 said albumen fully, thereby suppress the secretion of neurotransmitter.In the present invention; For the efficient that promotes in cell, to send; Through 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

VAMP2(SEQ ID NO：2)

MSAIAATAPP AAPAGEGGPP APPPNLTSV

KYWW 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

MA

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 suppressor factor 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 property of height and the nexin transduction domain (PTD) that can in skin, eyes etc., send efficiently; With the structural domain that can combine VAMP-2 of people source SNAP25 or the structural domain that can combine syntaxin/SNAP25 of VAMP-2; Promptly; Be used to suppress the peptide that the SNARE complex body forms, in neurocyte, said 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 said 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 " among this paper) of peptide that is connected with derived from human source SNAP-25 (being also referred to as " SBD " among this paper) or derived from human source VAMP-2 (be also referred to as among this paper " PTD-SBD or VBD ").The structure of said fusion polypeptide can be represented by following formula 1:

[formula 1]

Wherein, [] _PTDIt is self cell-penetrating property (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, l-arginine, Xie Ansuan, glycocoll and proline(Pro); N is the integer of 5-30; And the total amino acid above 30% is arginine residues.The instance 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 property PTD peptide and SBD/VBD peptide, and can improve the flowability of said 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 glycocoll, it is understandable that and to use various terminal to realize the object of the invention as joint.

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 through between SBD or VBD peptide and PTD peptide, inserting about 3 glycine residues, to promote to be delivered to and to be absorbed into the efficient in the cell.

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 through use, for example adopts solid phase synthesis process or uses the compound 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 said 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 said fusion polypeptide through making the amino acid generation condensation that is positioned at C-end and N-end.

Said solid phase synthesis is that the C-terminal from polypeptide begins, through the amino protected amino acid of a α is coupled on the suitable resin.Among this paper, the amino protected amino acid of said α is coupled on hydroxymethyl resin or the chloromethyl resin through ester bond.Use Fmoc (9-fluorenylmethyloxycarbonyl) as the amino blocking group of α, and the amino acid of Fmoc-protection can be purchased acquisition from Beadtec Company.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 glycocoll, Methionin or Stimulina by suitable group in use.In this compound 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.Said peptide can extract in TFA solution and is separated with diethyl ether, and can come purifying through performance liquid chromatography (HPLC).

Second method is to use biological method to prepare the method for said fusion polypeptide.This method may further comprise the steps: (a) with the gene clone of said fusion polypeptide in the expression vector pPET that can express particular peptide, the recombinant vectors that can express said 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 said fusion polypeptide can be easily derived from the disclosed aminoacid sequence of this paper by those skilled in the art.In addition; Be used for inserting the system of selection and the general technological detail file of the cloning vector of said base sequence, the recombinant expression vector that uses the clone technology preparation, the method for using recombinant vectors to transform said 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 said target fusion polypeptide and from the product that obtains, collected said target fusion polypeptide, be conspicuous for a person skilled in the art.

On the other hand, the invention provides the neurotransmitter inhibitor that comprises fusion polypeptide, said 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, said neurotransmitter inhibitor can be delivered to partial position through PTD, for example, and skin.

Fusion polypeptide of the present invention can be delivered to efficiently in the cell with local location in, thereby suppress the secretion of neurotransmitter effectively.

Description of drawings

Fig. 1 representes the mechanism of neurotransmitter secretion.

Fig. 2 representes the result according to the HPLC analysis of synthetic polypeptide of the present invention.

Fig. 3 representes the cleavage point diagram of PTD conjugate.

Fig. 4 representes the agar gel photo of PTD-SBD and PTD-VBD.

Fig. 5 representes the cell-penetrating efficient of PTD conjugate.

Fig. 6 representes through being the MV 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 representes 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 drum of a standard; In this reactor drum, add activatory Fmoc-G that mole number is 0.5mmol and Fmoc-R (Fmoc-G and Fmoc-R are the amino acid of first C-terminal of polypeptide to be synthesized), begin synthesizing of said 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 N (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 with DMF washing 10 minutes.

(2) separation of fusion polypeptide and purifying

After synthetic the completion, said polypeptide is placed the healthy and free from worry pipe (corning tube) of 50-mL, and be cooled to 0 ℃.Then, the mixture of TFA of zero(ppm) water and 8.25ml that will contain thioanisole (thianisol), the 0.5mL of the phenol of 0.75g, the 1 of 0.25ml (EDT), 0.25ml joins in the said pipe, at room temperature reacts 4 hours.After reaction is accomplished, 30ml is lower than 50 ℃ ether joins through filtering in the solution after said resin separated from said peptide, precipitate said peptide, use twice of ether washing precipitation then.The deposition of collecting is dry, be dissolved among the MeOH, and through 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 said peptide of wash-out of 100% acetonitrile+0.01%TFA).Acetonitrile is removed from said peptide, and said 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 said fusion polypeptide of coding, Hind III cleavage site that N-is terminal and the terminal BamH I cleavage site of C-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 said fusion polypeptide is attached on the proteic base sequence of coding high expression level; Use restriction enzyme HindIII to separate said 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 said fusion polypeptide of coding of above-mentioned preparation, has prepared recombinant expression vector like this.In Fig. 3, provided the schematic gene mapping of said expression vector.

(2) expression of the preparation of intestinal bacteria transformant and fusion polypeptide and purifying

Through 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 preparatory cultivation 12 hours.Then, preparatory 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 lec operon, substratum was hatched 8 hours, to induce the expression of said fusion polypeptide.4 ℃ down and with 5,000rpm removes centrifugal 20 minutes of substratum then supernatant and keeps deposition.Then, said 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, pH 8.0) 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 said deposition 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 said lysate, adds 2.5mL ²⁺(Qiagen cat#30230), stirs mixture 1 hour with 200rpm under 4 ℃, so that said fusion polypeptide is attached to Ni-NTA agar syrup ²⁺On-NTA the agarose.Then, with mixture through 0.8 * 4cm chromatographic column (BioRad, cat#731-1550).Damping fluid 2 (50mM NaH with 4mL ₂PO ₄, 300mM NaCl, the imidazoles of 20mM, pH 8.0) and wash the material twice that obtains, use damping fluid 3 (the 50mM NaH of 0.5mL then ₂PO ₄, 300mM NaCl, the imidazoles of 250mM, pH 8.0) wash four times, thereby collect said fusion polypeptide.The fusion polypeptide of collecting is carried out SDS-PAGE, and through examining the blue dyeing of Ma Shi, the result of analysis is 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 said fusion rotein, add 2mL with ice-cooled damping fluid (4M NH ₂OH, 0.4M K ₂CO ₃, 6M GuHCl, pH 9.0) make and contain 2M NH in the damping fluid that obtains ₂OH, 0.2M K ₂CO ₃, 3M GuHCl, pH 9.0.Then, under 45 ℃ with 225rpm stirred solution 5 hours so that said fusion rotein separates into fusion polypeptide and translocator, and through add 12N HCl with the pH regulator of said solution to 2-3, further stirred again 10 minutes.After reaction is accomplished, be 6.5 through adding 12.5N NaOH with the reaction soln pH that neutralizes, then through being filled with Ni ₂₊The post of-NTA agarose is to separate translocator from said solution.The solution that obtains is carried out gel-filtration, salt component is removed lyophilize then, thereby the fusion polypeptide of acquisition purifying from said 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.Active for relatively, SBD and VBD have been prepared two kinds of fragments respectively.Said fragment sample is following:

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 hydrogencarbonate; T75 (Nunc) solution of 15% horse serum and 2.5% foetal calf serum (FBS)) the PC12 cell (pheochromocytoma, ATCC CRL-1721) that thaws in, 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 deposition, mixture was hatched one day.Then, with 10 μ M Ca ²⁺Stimulate mixture, analyze the amount of excretory catecholamine through 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.Can find out that from table 1 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

Negative control: do not stimulate

Positive control: use Ca ²⁺Stimulate but do not make medicament

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, with fluorescent substance FITC (available from Sigma; Resorcinolphthalein 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 through 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 through suppressing neurotransmitter; Respectively botulic neurotoxin and said 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 saline water 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 through said 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 the 300-3000Hz place trap signal.Then, (Digidata1320, Axon Instruments Inc.) are input in the computingmachine, and said signal is represented with the form of ripple through signal acquiring system with filtering signal.

Analysis is stored in the signal in the computingmachine, and wave amplitude (dY) is for through with the frequency being negative peak and the value between the posivtive spike of the average potential of the CMAPs that obtains of 2Hz repetitious stimulation 50 times; Time is the time course (tC) (referring to Fig. 6) between the time point of time point and negative peak of electricity irritation.

Of Fig. 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; Under the prerequisite that does not depart from disclosed scope of the present invention and spirit in accompanying claims, can carry out various changes, increase and replacement.

Industrial applicibility

As stated, fusion polypeptide of the present invention can be delivered in the cell and local location effectively, thereby suppresses the secretion of neurotransmitter effectively.

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

Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg

1 5 10 15

Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met

20 25 30

Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val

35 40 45

Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met

50 55 60

Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp

65 70 75 80

Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys

85 90 95

Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val

100 105 110

Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala

115 120 125

Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn

130 135 140

Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu

145 150 155 160

Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg

165 170 175

Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala Asn Lys Thr Arg Ile

180 185 190

Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly

195 200 205

<210>2

<211>117

<212>PRT

< 213>artificial sequence

<220>

<223>VAMP2

<400>2

Met Ser Ala Thr Ala Ala Thr Ala Pro Pro Ala Ala Pro Ala Gly Glu

1 5 10 15

Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Val Asn Arg Arg

20 25 30

Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg

35 40 45

Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu

50 55 60

Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr

65 70 75 80

Ser Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met

85 90 95

Met Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile

100 105 110

Val Tyr Phe Ser Ser

115

<210>3

<211>288

<212>PRT

< 213>artificial sequence

<220>

< 223>syntaxin

<400>3

Met Lys Asp Arg Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp

1 5 10 15

Asp Asp Asp Val Thr Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu

20 25 30

Phe Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala

35 40 45

Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser

50 55 60

Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser

65 70 75 80

Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile

85 90 95

Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Leu

100 105 110

Asp Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val

115 120 125

Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg Glu Arg

130 135 140

Cys Lys Gly Arg Ile Lys Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr

145 150 155 160

Thr Ser Glu Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile

165 170 175

Phe Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu

180 185 190

Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser

195 200 205

Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu

210 215 220

Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala

225 230 235 240

Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys

245 250 255

Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile Ile Cys Cys

260 265 270

Val Ile Leu Gly Ile Ile Ile Ala Ser Thr Ile Gly Gly Ile Phe Gly

275 280 285

<210>4

<211>37

<212>PRT

< 213>artificial sequence

<220>

<223>SBD

<400>4

Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala

1 5 10 15

Gln Ala Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys

20 25 30

Met Leu Gly Ser Gly

35

<210>5

<211>58

<212>PRT

< 213>artificial sequence

<220>

<223>VBD

<400>5

Asn Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp

1 5 10 15

Ile Met Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu

20 25 30

Ser Glu Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln

35 40 45

Phe Glu Thr Ser Ala Ala Lys Leu Lys Arg

50 55

<210>6

<211>11

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1

<400>6

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg

1 5 10

<210>7

<211>9

<212>PRT

< 213>artificial sequence

<220>

<223>Sim-2

<400>7

Ala Lys Ala Ala Arg Gln Ala Ala Arg

1 5

<210>8

<211>11

<212>PRT

< 213>artificial sequence

<220>

<223>Tat

<400>8

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210>9

<211>16

<212>PRT

< 213>artificial sequence

<220>

<223>Antp

<400>9

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>10

<211>34

<212>PRT

< 213>artificial sequence

<220>

<223>VP22

<400>10

Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr

1 5 10 15

Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro

20 25 30

Val Glu

<210>11

<211>7

<212>PRT

< 213>artificial sequence

<220>

<223>R7

<400>11

Arg Arg Arg Arg Arg Arg Arg

1 5

<210>12

<211>26

<212>PRT

< 213>artificial sequence

<220>

<223>MTS

<400>12

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Ala Ala Ala Asp Gln Asn Gln Leu Met Pro

20 25

<210>13

<211>21

<212>PRT

< 213>artificial sequence

<220>

<223>pep-1

<400>13

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>14

<211>51

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-SBD

<400>14

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Glu Ile

1 5 10 15

Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala

20 25 30

Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu

35 40 45

Gly Ser Gly

50

<210>15

<211>72

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-VBD

<400>15

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Asn Arg

1 5 10 15

Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met

20 25 30

Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu

35 40 45

Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu

50 55 60

Thr Ser Ala Ala Lys Leu Lys Arg

65 70

<210>16

<211>51

<212>PRT

< 213>artificial sequence

<220>

<223>Tat-GGG-SBD

<400>16

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Gly Glu Ile

1 5 10 15

Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala

20 25 30

Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu

35 40 45

Gly Ser Gly

50

<210>17

<211>40

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-SBDF1

<400>17

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Ile Met

1 5 10 15

Glu Lys Ala Gln Ala Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg

20 25 30

Ala Thr Lys Met Leu Gly Ser Gly

35 40

<210>18

<211>28

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-SBDF2

<400>18

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Lys Thr

1 5 10 15

Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met

20 25

<210>19

<211>45

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-VDBF1

<400>19

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Asn Arg

1 5 10 15

Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met

20 25 30

Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys

35 40 45

<210>20

<211>41

<212>PRT

< 213>artificial sequence

<220>

<223>Hph-1-GGG-VBDF2

<400>20

Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Leu Ser

1 5 10 15

Glu Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe

20 25 30

Glu Thr Ser Ala Ala Lys Leu Lys Arg

35 40

Claims

1. fusion polypeptide that is used for suppressing in vivo neurotransmitter secretion, the aminoacid sequence of this fusion polypeptide is SEQ ID NO:14 or SEQ ID NO:17.

2. compsn that is used to suppress neurotransmitter secretion, said composition contains the described fusion polypeptide of claim 1 as activeconstituents.