JP2010502592A - Fusion peptide for blocking neurotransmitter and method for transmitting the same - Google Patents

Abstract

  In order to suppress the secretion of a neurotransmitter, the present invention fuses a polypeptide at the binding site of three proteins constituting a SNARE complex with a protein transduction domain (PTD) in a neuron. The present invention relates to a method for improving intracellular transmission efficiency and transmission to a local site.

Description

  In order to suppress the secretion of a neurotransmitter, the present invention fuses a polypeptide at the binding site of three proteins constituting a SNARE complex with a protein transduction domain (PTD) in a neuron. The present invention relates to a method for improving intracellular transmission efficiency and transmission to a local site.

For the secretion of neurotransmitters, synaptobrevin (Vesicle-associate membrane protein, VAMP) present on the surface of vesicles carrying neurotransmitters, pre-synaptic Three proteins, SNAP25 and Syntaxin, present in the plasma membrane) form a SNARE complex and are secreted extracellularly by Ca ²⁺ stimulation (see FIG. 1).

  The most commonly used botulinum neurotoxins as neurotransmitter secretion inhibitors have sub-types such as A, B, C, D, E, and F. These are VAMP-2, SNAP25, a specific site of syntaxin, is cleaved to prevent the formation of SNARE complex, thereby inhibiting neurotransmitter secretion (Non-patent Document 1).

  Since such botulinum neurotoxins are very toxic, as described above, research results on the suppression of neurotransmitter secretion using peptide fragments cleaved by botulinum neurotoxins have been reported. Since only a part of the entire protein is used, it is generally less specific and less active than botulinum neurotoxin. Also, when the peptide size increases, the rate of transmission into the cell increases. Since it is slow, the intracellular transmission efficiency is actually lower than that of botulinum neurotoxin that binds to the receptor and is induced in the cell (Non-patent Document 2).

  A general method for biologically and effectively transmitting macromolecules having biological activity without damaging cells both inside and outside the living body is required (Non-patent Document 3). Examples of such methods include chemical addition of lipid peptides (Non-Patent Document 4) or methods using a basic polymer such as polylysine and polyarginine (Non-Patent Document 5). Has not yet been verified. The fact that folic acid (Non-patent Document 6) used as a transporter moves into cells as a folic acid-salt conjugate has been reported, but it has not yet been confirmed whether it is transmitted into the cytoplasm. Pseudomonas exotoxin is also used as a kind of transporter (Non-patent Document 7). However, even in these methods, it is not clear about the effect on the movement of biologically activated 'transmitted' substance into cells and its general applicability. Therefore, there is a continuous need for a method that can safely and effectively transmit a biologically active substance into the cytoplasm and nucleus of living cells.

  PTD is one of the results presented as a result of research on such requirements, and the most researched among them was the transcription of human immunodeficiency virus-1 (HIV-1). It is a Tat protein that is a factor. When this protein passes through the cell membrane, it is from a portion of the 47th to 57th amino acids (YGRKKRRQRRR) in which positively charged amino acids are concentratedly distributed, rather than in the complete form of 86 amino acids. It became clear that it is more effective when it is the form which becomes (nonpatent literature 8). As other examples in which the effect as PTD was confirmed in this way, the 267th to 300th amino acids of the VP22 protein of HSV-1 (Herpes Simplex Virus type 1) (Non-patent Document 9), and Drosophila ANTP (Antennapedia) There are 339th to 355th amino acids of protein (Non-patent Document 10), and the effect was also confirmed in the case of artificial peptides enumerated electrically positive amino acids (Non-patent Document). 11).

Schiavo et al. Nature 359, 832-835, 1992 Antonio V. Ferror-Monitiel et al. FEBS letters, 435, 84-88, 1998 L.A. Sternson, Ann. N.Y. Acad. Sci., 57, 19-21 (1987) P. Hoffmann et al. Immunobiol., 177, 158-170 (1998) W-C. Chen et al., Proc. Natl. Acad. Sci., USA, 75, 1872-1876 (1978) C.P. Leamon and Low, Proc. Natl. Acd. Sci., USA, 88, 5572-5576 (1991) T. I. Prior et al., Cell, 64, 1017-1023 (1991) Fawell S. et al. Proc. Natl. Acad. Sci. USA 91, 664-668 (1994) Elliott G. et al. Cell, 88, 223-233 (1997) Schwarze S. R. et al. Trends Pharmacol Sci. 21, 45-48 (2000) Laus R. et al. Nature Biotechnol. 18, 1269-1272 (2000)

  Therefore, the present invention uses a domain in which three kinds of proteins forming a SNARE complex are mutually coupled to increase the efficiency of transmission into cells and competitively interfere with protein binding, thereby causing neurotransmitters. It aims at suppressing the secretion.

  In the present invention, in order to increase the efficiency of transduction into cells, a fusion protein is produced by conjugating a protein transmitter (PTD, Protein Transduction Domain) and binding domains of three kinds of proteins forming a SNARE complex. A peptide (fusion polypeptide) is produced and used to suppress the secretion of neurotransmitters.

  The amino acid sequences of synaptobrevin (VAMP-2), SNAP25, and syntaxin constituting the SNARE complex are as follows.

  In SNAP25 (SEQ ID NO .: 1), the 170th amino acid to the 206th amino acid (the part indicated by bold letters and underline) are the parts that bind to VAMP-2 (SEQ ID NO .: 4, SBD), VAMP-2 (SEQ ID NO .: 2), amino acids 29 to 86 (indicated by bold letters and underlined) are domains (SEQ ID NO. : 5, VBD). In the present invention, PTD is conjugated to each of SBD (SEQ ID NO .: 4) or VBD (SEQ ID NO .: 5) and used as a neurotransmitter secretion inhibitor.

  To achieve the above object, the present invention provides a domain in which human SNAP25 binds to VAMP-2, and a domain in which VAMP-2 binds to syntaxin, SNAP25, ie, neurotransmitter secretion in neurons. Provided is a fusion polypeptide of a peptide that suppresses the structure of the SNARE complex and a protein transduction domain (PTD, Protein Transduction Domain) having high cell permeability and transmission efficiency to the skin, eyes, and the like. The fusion peptide according to the present invention is characterized in that it inhibits the secretion of neurotransmitters such as catecholamine and acetylcholine, greatly increases the inhibitory effect on the secretion of neurotransmitters by the binding of PTD, and the cytotoxicity is not changed.

  The fusion peptide according to the present invention is a fusion polypeptide in which a PTD peptide is bound to a peptide derived from human SNAP-25 (referred to as 'SBD') and a peptide derived from human VAMP-2 (referred to as 'VBD'). With regard to (sometimes referred to herein as 'PTD-SBD or VBD'), the chemical formula of the peptide sequence is

In the above formula, [] _PTD is an autologous cell-penetrating PTD peptide, R ₁ is one or more selected from the group consisting of tylosin, alanine, arginine, valine, glycine and proline side chain, and n is It is an integer of 5 to 30, and 30% or more of all amino acids is arginine. Examples of PTD include Hph-1 (SEQ ID. NO .: 6-YARVRRRGPRR), Sim-2 (SEQ ID. NO .: 7-AKAARQAAR), Tat (SEQ ID. NO .: 8-YGRKKRRQRRR), Antp (SEQ ID. NO .: 9-RQIKIWFQNRRMKWKK), VP22 (SEQ ID. NO .: 10-DAATATRGRSAASRPTERPRAPARSAS RPRRPVE), R7 (SEQ ID. NO .: 11-RRRRRRR), MTS (SEQ ID. NO .: 12-AAVALLPAVLLALLAPAAADQNQLMP ), Pep-1 (SEQ ID. NO. 13-KETWWETWWTEWSQPKKKRKV). [] _Gly links the autologous cell-penetrating PTD peptide and SBD or VBD peptide, enhances the fluidity of the fusion peptide, and the binding efficiency between the permeated PTD-SBD or VBD and SNAPE complex To improve. m is an integer of 0-5. In Formula 1 above, it is suggested that Gly was used as the linker, but various linkers can be used for the purposes of the present invention.

  SBD (SEQ ID. NO .: 4) and VBD (SEQ ID. NO .: 5) are peptides derived from human SNAP-25 and VAMP-2, respectively, and the present invention includes sections of these domains. It will be understood by those skilled in the art that any of the sequences of SEQ ID NOs: 4 and 5 which have been subjected to arbitrary modifications, that is, substitutions, insertions and deletions can be used as the SBD and VBD portions of the fusion peptide according to the present invention. It will be.

  According to the present invention, by inserting about 3 glycine residues between the SBD or VBD and the PTD peptide, the efficiency of transmission and absorption into the cell can be increased.

  On the other hand, the method for producing the fusion peptide according to the present invention may be a method clearly known in the art, for example, a solid phase synthesis method or a synthesis method using a recombinant expression system. However, the method for producing a fusion peptide according to the present invention is not limited to these methods.

  First, a pure fusion peptide can be synthesized using an organic synthesizer for peptide synthesis. This method is Merrifield's solid phase peptide synthesis method (J. Am. Chem. Soc. 85, 2149-21, 54 (1963)), and a peptide semi-automatic synthesizer (Peti-Syzer Model PSS-510). ) To condense monomers having reactivity at the C-terminal and N-terminal of amino acids to synthesize fusion peptides.

  Solid phase synthesis begins by coupling an α-amino group protected amino acid from the carboxyl terminal amino acid to a suitable resin. At this time, an amino acid in which the α-amino group is protected is attached to the hydroxymethyl resin or chloromethylated resin by an ester bond. As a group for protecting the α-amino group, Fmoc (9-fluorenylmethoxycarbonyl) is used (amino acids protected by Fmoc can be purchased through BIDTECH). For example, the amino group of Arg is reactive. Reactive residues of Gly, Lys, and Gln are protected by appropriate groups such as tert-butyl (t-Bu) and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl). Utilized Fmoc-amino acids. Peptide synthesis proceeds by sequentially removing the amino-terminal protecting group of the peptide chain attached to the solid support resin with a reagent such as trifluoroacetic acid (TFA) and phenol. Peptides can be extracted from TFA solution using diethyl ether, separated and purified by high performance liquid chromatography (HPLC).

  Secondly, a method for producing a fusion peptide using a biological method is as follows: (a) cloning the gene of the fusion peptide into an expression vector pPET capable of expressing a specific peptide and expressing it in the form of the fusion peptide. And (b) mass expression in the form of a recombinant transporter-PTD-SBD (VBD) peptide in E. coli using the pPET-PTD-SBD (VBD) expression vector. Followed by pure separation and purification. At this time, the base sequence encoding the fusion peptide for producing the recombinant vector can be easily inferred by those skilled in the art from the amino acid sequence disclosed in the present application, and cloning for inserting such base sequence is performed. Vectors, recombinant expression vectors produced using cloning techniques, host cells for transforming with this recombinant vector to express the fusion peptide of interest, methods for transforming the host with recombinant vectors, transformed Those skilled in the art can easily recognize selections and general technical matters regarding a method for expressing the target fusion peptide from the host cell and a method for obtaining the target fusion peptide from the final product.

  On the other hand, the present invention provides a neurotransmitter inhibitor comprising a fusion peptide in which the protein transmitter PTD peptide is bound to a human-derived SBD or VBD peptide.

  Such a neurotransmitter inhibitor can be applied to various fields such as pain relief, wrinkle improvement, and quadrangular jaw improvement, and in particular, can be transmitted to a local site such as skin by PTD.

  The fusion peptide according to the present invention is excellent in intracellular transmission efficiency and transmission efficiency to a local site, and therefore effectively suppresses the secretion of neurotransmitters by the fusion peptide.

It is the figure which showed the secretion mechanism of the neurotransmitter. The HPLC result of a synthetic peptide is shown. It is a cleavage map of DNA of a PTD conjugate. It is an agarose gel photograph of PTD-SBD and PTD-VBD. The cell permeation efficiency by PTD conjugation is shown. The average of composite muscle action potentials recorded by stimulating the sciatic nerve 50 times at 2 Hz, the amplitude of action potential (dY) indicating the amount of muscle contraction, and the delay time until the generation of the muscle action potential after stimulation. (TC) is shown. It shows the amplitude of compound muscle action potential (Amplitude of compound muscle action potential).

  Examples of the invention will be described below.

Example 1: Production of PTD-SBD (VBD) conjugate (1) Production of PTD-SBD (VBD) conjugate
YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG (PTD-GGG-SBD polypeptide)
A fusion polypeptide having the amino acid sequence of YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR (PTD-GGG-VBD polypeptide) was synthesized by a solid phase peptide synthesis method using a peptide semi-automatic synthesizer (Peti-Syzer Model PSS-510). Put 0.1 mmol of Rink Amide MBHA resin in a standard reaction vessel, and put activated 0.5 mmol of Fmoc-G and Fmoc-R, respectively, which is the first amino acid at the C-terminal of the peptide to be synthesized. I started synthesis. The amino acid residues corresponding to the sequence order from the C-terminal amino acid to the N-terminal amino acid were reacted with 0.5 mmol three times. Fmoc deprotection was performed 3 times for 10 minutes using 20% piperidine diluted with dimethylformamide (DMF), washed for 10 minutes with DMF, and coupled for 100 minutes. For coupling, a system using N-hydroxybenzotriazole (HOBT) and 1,3-diisopropylcarbodiimide (DIC) and washing with DMF for 10 minutes was used.

(2) After completion of separation and purification synthesis of the fusion peptide , 50 mL of the peptide was put into a corning tube and cooled to 0 ° C., and then 0.75 g of phenol, 1,2-ethanedithiol (EDT) A cleavage solution having a mixing ratio of 0.25 mL, thianisol 0.25 mL, distilled water 0.5 mL, and TFA 8.25 mL was added, followed by reaction at room temperature for 4 hours. After the reaction was completed, the peptide and the separated resin were filtered, and 30 mL of diethyl ether at 50 ° C. or lower was added to the remaining solution to precipitate the peptide, and diethyl ether was further added twice and washed. The obtained precipitate was dried, dissolved in MeOH, and purified by HPLC. The columns used for purification were C18 analysis column 218TP54 (VyDac) and C18 fractionation column 218TP1022 (VyDac). The purification conditions were 1 mL / min and 25 mL / min, respectively, and the peptide was eluted using A: 100% H ₂ O + 0.01% TFA and B: 100% acetonitrile + 0.01% TFA as solvents. Acetonitrile was removed from the eluted peptide and lyophilized to obtain a highly pure peptide (see FIG. 2).

Example 2 Production and purification of fusion peptide expression vector using microbial expression system (1) Production of expression vector For the production of a base sequence encoding the fusion peptide, Hind III was added to the N-terminus of pUC19 (source: invitrogen), A mold was made with BamH I at the C-terminus. In order to combine the poly His tag region for high expression and easy purification of the fusion peptide with the base sequence encoding the highly expressed protein, the template was separated through the reaction of restriction enzymes Hind III and BamHI, and the Oiaquick purification kit Was purified using. The expression vector and pPET vector were treated with restriction enzymes Hind III and BamHI, and purified using an Oiaquick purification kit. The purified expression vector was cloned using the nucleotide sequence template encoding the prepared fusion peptide to produce a recombinant expression vector. A schematic gene map of the expression vector is as shown in FIG.

(2) Production of E. coli transformant and expression and purification of fusion peptide Escherichia coli BL21 (source: invitrogen) is transformed by the heat shock transformation method (Heat shock transformation) using the expression vector produced above. Then, 1 mL of transformed E. coli was inoculated at 1% in 100 mL of LB medium and pre-cultured with stirring at 37 ° C. for 12 hours. Subsequently, each of these was again inoculated into 1,000 mL of LB medium and cultured at 37 ° C. for 4 hours, and then 1 mM IPTG (isopropyl β-D-thiogalactopyranoside, GibcoBRL cat. # 15529-019) was added. It was added to induce the expression of the lac operon and cultured for 8 hours to induce the expression of the fusion peptide. The above culture broth was centrifuged at 5,000 rpm for 20 minutes at 4 ° C. to leave only the pellet, and after removing the supernatant, 10 mL of 1 mg / mL lysozyme (Sigma, cat, # L-7651) was contained. Dissolve the pellet in a buffer solution (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0), leave it on ice for 30 minutes, then use an ultrasonic grinder (Heat systems, ultrasonic processor XL). The process of injecting ultrasonic waves for 10 seconds at an intensity of 300 W and cooling for 10 seconds was repeated until the cumulative ultrasonic injection time was 3 minutes. The eluate was centrifuged at 12,000 rpm for 20 minutes at 4 ° C. to remove E. coli debris and only the pure eluate was separated. Add 2.5 mL of 50% Ni ²⁺ -NTA agarose slurry (Qiagen, cat # 30230) to the separated eluate and stir at 4 ° C. and 200 rpm for 1 hour to bind the fusion protein and Ni ²⁺ -NTA agarose. The mixture was poured onto a chromatographic 0.8 × 4 cm column (BioRad, cat # 731-1550). After washing twice with 4 mL of buffer solution 2 (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0), 0.5 mL of buffer solution 3 (50 mM NaH ₂ PO ₄ , 300 mM NaCl, The fusion protein was fractionated 4 times with 250 mM imidazole (pH 8.0) and subjected to SDS-PAGE, followed by confirmation by Coomassie blue staining. The results are shown in FIG. In FIG. 4, lane 1 is a standard molecular weight protein, lane 2 is PTD-SBD, and lane 3 is PTD-VBD. 2 mL of ice-cold 4M NH ₂ OH, 0.4M K ₂ CO ₃ , 6M GuHCl, pH 9.0 is added to the fusion protein to give a final buffer solution concentration of 2M NH ₂ OH, 0.2M K ₂ CO _{3. After} adjusting to 3M GuHCl, pH 9.0, stir at 45 ° C. and 225 rpm for 5 hours to separate the fusion protein into the fusion peptide and transporter protein, and add 12N HCl to adjust the pH to 2-3. And further stirred for 10 minutes to complete the reaction. After 12.5N NaOH was added to neutralize to pH 6.5, the transporter protein was separated in a packed Ni ₂₊ -NTA agarose column and then gel filtered to obtain a buffer solution. After removing the salt component, lyophilization gave a pure fusion peptide.

Example 3 Inhibition effect of neurotransmitter secretion by fusion peptide in experimental tube The secretion inhibitory effect of neurotransmitter of the fusion peptide produced in Examples 1 and 2 was analyzed. Two sections of SBD and VBD were prepared and the activities were compared.

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
PC12 cells (Pheochromocytoma, ATCC CRL-1721) in T75 (Nunc) with 15 mL of 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 15% horse serum, and 2.5% FBS. After thawing with F-12K medium (Gibco) and culturing for 2 days to obtain a cell density of 625,000 cells / cm ² , the supernatant was removed by centrifugation to remove the same volume. F-12K medium and drug were each added and cultured for 1 day, stimulated with 10 uM Ca ²⁺ , and the amount of secreted catecholamine was analyzed through fluorescent HPLC. The drug concentration was ^10-6 to ^10-5 (M). The results are shown in Table 2. From Table 2, it was found that the compound represented by [Chemical Formula 1] used in the present invention is excellent in the neurotransmitter secretion inhibitory effect.

Example 4: Intracellular transmission effect due to PTD conjugation In order to analyze the intracellular transmission effect due to the binding of PTD, each of SBDF1 peptide conjugated with PTD and SBDF1 peptide unconjugated to PTD, FITC (source: Sigma, Fluorescein 5-isothiocyanate, 27072-45-3), which is a fluorescent substance, was labeled and analyzed by Facs (FACSCalibur, Becton Dickinson) (see FIG. 5). As a result, it was analyzed that PTD conjugation improved the transmission effect about 50 times.

Example 5: Test of muscular paralysis with PTD conjugate Botulinum neurotoxin and peptide according to the present invention block neurotransmitters and cause muscle paralysis, so botulinum neurotoxin and peptide were injected into rat anterior tibial muscle. After 1 week, 2 weeks, and 3 weeks, the amplitude (dY) of the compound muscle action potential (CMAP) of the anterior tibial muscle by electrical stimulation of the sciatic nerve was measured. First, 8 weeks old SD rat (supplier: HYOCHANG) was transferred to botulinum neurotoxin A (manufacturer: allergan, supplier: Sigma) and samples 1, 3 and 6 on the right anterior tibialis muscle of the hind paw. 5 μl each was injected. The botulinum neurotoxin was injected by dissolving 400 U of botulinum neurotoxin in 1 mL of saline to make a 400 U / mL solution, and 5 μl was injected once into the rat. Samples 1, 3, and 6 were administered at 10 ng, 100 ng, and 500 ng per rat, respectively.

  Compound muscle action potential was measured by anesthetizing rats by intraperitoneal injection of ketamine / xylazine cocktail, and then placing the rats on their prone table on an examination table maintained at 30 ° C. and fixing their limbs. The hip joint, knee joint, and ankle joint of the lower limb to be examined were each bent and fixed at 90 degrees, and the recording electrode, the reference electrode, and the ground electrode were attached to the anterior tibial muscle, the radial tendon, and the back side of the foot, respectively. A stimulation electrode (+) needle was inserted into the popliteal region, and a stimulation electrode (−) needle was inserted into the pubiceal muscle of the posterior pubic and proximal femurs. The compound muscle action potential of the anterior tibial muscle was induced by stimulating the sciatic nerve by passing an electric current through the stimulation electrode in the range of 1 to 5 microA. Compound muscle action potentials were repeatedly induced by 50 repeated stimulations at 2 Hz at about 150% intensity of the stimulation exhibiting the maximum amplitude. After resting for 5 minutes, compound muscle action potentials were repeatedly induced by repeated stimulation at 20 Hz 50 times. These signals are amplified 500 times with a bioelectric amplifier and signal processor (CyberAmp380, Axon Instruments, Inc.) and filtered in the range of 300 to 3000 Hz, and then a signal acquisition device (Digidata1320, Axon Instruments, Inc.) is used. And input it to a computer to show the waveform.

  Analyzing the signal stored in the computer and repeating 50 times at 2 Hz, the difference in amplitude (dY) between the negative peak and the positive peak of the average potential of CMAP, electrical stimulation, and the time delay (tC) between negative peaks Was calculated (see FIG. 6).

  As shown in FIG. 7, in the case of botulinum neurotoxin and sample 1, sample 3 and sample 6, the result of the combined muscle action potential is shown in FIG. In the case of sample 3 and sample 6, the effect was the same as that of the botulinum neurotoxin as the dose of the substance increased.

  Although the present invention has been described with reference to the preferred embodiments, those skilled in the art will recognize that the invention is within the scope and spirit of the invention as defined by the following claims. It will be understood that various modifications and changes can be made.

  As described above, the fusion peptide according to the present invention is excellent in intracellular transmission efficiency and transmission efficiency to a local site, and thus effectively suppresses secretion of a neurotransmitter by the fusion peptide.

Claims (5)

  A protein transduction domain (PTD) consisting of 5 to 30 amino acids and containing 30% or more of arginine, and a binding domain of SNAP25 (SEQ ID. NO .: 4) or VAMP-2 constituting SNARE complex A fusion polypeptide for inhibiting secretion of a neurotransmitter in vivo, conjugated with a binding domain (SEQ ID. NO .: 5).   The protein carrier PTD has any one amino acid sequence selected from the group consisting of SEQ ID NO .: 6 to SEQ ID NO .: 12, and SEQ ID NO .: 13. A fusion polypeptide according to 1.   A protein carrier is conjugated with a SNAP25 binding domain (SEQ ID. NO .: 4) or a VAMP-2 binding domain (SEQ ID. NO .: 5) by a linker. The fusion polypeptide according to claim 1 or 2.   The fusion polypeptide according to claim 3, wherein the linker consists of three glycines.   A composition for inhibiting neurotransmitter secretion, comprising the fusion peptide according to claim 1 or 2 as an active ingredient.

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