CN109369785A - Ccotx1 toxin and its application - Google Patents

Abstract

The invention mainly relates to Ceratotoxin-1 (Ccotx1) toxin and its applications, the polypeptide toxin can effectively and selectively block Nav1.7 (voltage-gated sodium channel α subunit families), therefore can alternatively property Na-ion channel blocker and in the molecule mechanism of related Nav1.7 sodium-ion channel, nerve signal transmitting, disease treatment etc. have many applications.

Description

Ccotx1 toxin and its application

Technical field

Purposes more particularly to one kind the present invention relates to peptide material and its in drug preparation can be effectively and selective Ground blocks the polypeptide toxin and application thereof of Nav1.7 (voltage-gated sodium channel α subunit families).

Background technique

Voltage-gated sodium channels (VGSCs) are widely distributed, either vertebrate or invertebrate In various tissues, there is expression in these channels.VGSCs is played in the generation and outburst of action potential and Neurotransmission Important function.Many diseases and Voltage-gated sodium channels are closely related, such as pain, epilepsy etc..

VGSCs is a kind of high molecular weight on excitable cells film, the glycoprotein of multiple cross-film.2003, international pharmacology Federation discloses the naming rule of VGSCs.English abbreviation (NaV) indicates voltage-sensitive sodium-ion channel, and wherein Na represents sodium Ion channel, V then indicate voltage-sensitive channel.Presently found nine kinds of sodium-ion channels are respectively designated as Navl.l- Nav1.9, " 1 " is for indicating 1 type subfamily, and " 1 " subsequent number then represents specific hypotype.The ammonia of nine kinds of sodium channel hypotypes Base sequence difference is analysis shows that nine kinds of hypotypes of Navl.l to Nav 1.9 are very conservative in the process in natural evolution, together The equal similarity of sequence is 75% or more between the sodium channel hypotype of one species, it means that the divergent sequences in sodium channel are then very The difference of the dynamics and physiological function between different subtype is determined in big degree.According to VGSCs to tetraodotoxin (TTX) Whether sensitive, nine kinds of sodium channel hypotypes are divided into tetraodotoxin responsive type and tetraodotoxin non-sensitive type again.The former includes Navl.l,Nav 1.2,Nav 1.3,Nav 1.4,Navl.6,Nav1.7；The latter mainly has Nav 1.5, Nav 1.8, Nav 1.9。

VGSCs is widely distributed in tissue body.It is found in the tissue such as central nervous system, heart, skeletal muscle Their expression, in addition to Nav 1.4 and Nav 1.5, all sodium, which leads to hypotype, to be had on people's dorsal root ganglion (DRG) cell Expression；Nav 1.1, Nav 1.3, Nav 1.6 are also distributed in the transverse tube of heart tissue.They show certain again simultaneously Tissue specificity, if Voltage-gated sodium channels Nav1.5 is mainly distributed on the cell membrane of cardiac muscle cell, and Nav 1.4 is only It is distributed only on Skeletal Muscle Cell, Nav1.6 is on alveolar smooth muscle cell.

Nav1.7 is mainly expressed in periphery sympathetic neuron and sensory neuron.Nav1.7 accumulates in nerve fibre end End amplifies small-scale threshold depolarization, and as the excitatoty threshold value channel of adjusting.Nav1.7 function and various pain status Related including acute, inflammatory and/or neuropathic pain.In human body, it has been found that the function gain mutation of Nav1.7 with it is primary Property acromelalgia (disease is characterized in that limbs cusalgia and inflammation) is related to paroxysmal severe pain disease.It is consistent with this observation It is that non-selective sodium channel inhibitor lidocaine, mexiletine and carbamazepine can be relieved the symptom of these pain diseases.In people In vivo, the mutation of the function of the Nav1.7 property lost leads to rare autosomal recessive disease congenital absence of pain, it is characterised in that Complete missing is insensitive to the perception of pain stimulation or to pain stimulation.

A variety of polypeptide toxins with the interaction of voltage-sensitive sodium channel are had now been found that.Pass through acting on for selectivity The some regions of sodium channel, polypeptide toxin or delay channel inactivation, or accelerate channel activation, or to block sodium current not change but logical Road dynamic characteristic.There are 6 regions to be proved in the interaction of channel and toxin on Voltage-gated sodium channels It plays an important role.Sandrine Cestele etc. is named these regions, is followed successively by 1~site of site 6, and will make It is referred to as the site toxin for a certain site toxin.These sites are mainly distributed on the outside of cell membrane, transmembrane segment and duct Inside.For example, site 1 is made of two cyclic structures, it is located at sodium channel duct area.The toxin for acting on the region mainly has river Tetrodotoxin, conotoxin and ciguatoxin.2, site is made of two cross-film sections of DI-S6 and DIV-S6 in α subunit, The effects of frog skin toxin, aconitine, is in the region.δ-A CTX, sPhTx2, the polypeptide toxins such as δ-atracotoxins act on position Point 3, the region are located at the extracellular connection ring of S3~S4 on the α subunit DIV of sodium channel, and the used time is mutually done in polypeptide toxin and channel can be with It prevents the movement of DIV-S4 voltage sensitive element and changes the coupling of channel activation and inactivation, to inhibit channel current, make Be with the characteristics of mode: polypeptide toxin can delay channel inactivation after being integrated to site 3, but not influence channel activation.δ- Atracotoxins is typical 3 polypeptide toxin of sodium channel site, it can inhibit sodium current and delay the inactivation of sodium channel. SPhTx2 not only delayed sodium channel to inactivate as doubtful 3 toxin of site, the toxin, but also the activation of channel stable state and stable state can be made to lose It is living to drift about toward direction of depolarization.For δ-atracotoxins compared with traditional 3 toxin α of site-scorpion venom, they are mutual with channel Mechanism of action respectively has the similarities and differences.Both toxin can delay channel inactivation, the Lys on toxin³、Arg⁵And Asp¹³With sodium channel It plays a crucial role in interaction, the Key residues on sodium channel with toxin interaction are all Glu¹⁶¹³、Glu1⁶¹⁶And Lys¹⁶¹⁷, but α-scorpion venom will increase sodium channel current, and δ-atracotoxins then reduces sodium current.Toxin binding site 4 is main It is two extracellular rings connection between the S1-S2 on α subunit DII and between S3-S4, the polypeptides poison such as HWTX-IV, JZTX-III Element acts on site 4, inhibits to Preference 1.7 sodium current of Nav, sodium channel when by capturing in quiescent condition Domain II voltage sensitive element inhibits sodium current.Compared with traditional 4 toxin β of site-scorpion venom, the phase of they and sodium channel There are larger differences for interaction mechanism.HWTX-IV does not influence the activation of Nav 1.7, and JZTX-III makes the activation voltage in channel to going Polarization direction drift, and β-scorpion venom makes the activation voltage in channel drift about to hyperpolarization.In addition, the former is capture in tranquillization shape The voltage sensitive element of state, and the latter is the voltage sensitive element that capture is in open state.

Sodium channel inhibitor used at present, for example, carbamazepine, lidocaine and aforementioned polypeptides toxin, exist Multiple industry types of non-selective batrachotoxin, easily cause cental system, circulatory system side effect (such as dizziness, incoordination, Insomnia, nausea and headache etc.) defect, or have the defects that validity is low, safety is low, thus the clinic for limiting them is answered Be widely used.

Summary of the invention

As noted previously, as the drug binding site of each sodium channel is very conservative, and even adjacent sequences are also very much like, mesh Preceding 1.7 specific inhibition agent of Nav generally acknowledged not yet.One of the objects of the present invention is to provide Ccotx1 toxin, especially mention There is the polypeptide toxin of high selectivity and efficient functionality for a kind of couple of Nav 1.7.

The specific technical solution of the present invention is as follows:

Ccotx1 toxin, the polypeptide toxin include following sequence:

X₁CLGX₂FX₃X₄CX₅PX₆NDKCCX₇X₈X₉X₁₀CSX₁₁X₁₂X₁₃X₁₄WCKX₁₅X₁₆L (SEQ ID NO:2), in which:

X₁For D, I or Z；

X₅For A, I or M；

X₇For K or A；

X₈For S；

X₁₀For D；

X₁₂For E or K；

X₁₈For Y or K；

X₁₉For R or N；

X₂₀For L or Y；

X₂₁For V or T；

X₂₄For K or R；

X₂₅For S or R；

X₂₆For H or D；

X₂₇For N or R；

X₃₁For W or Y；

X₃₂For K or D.

As further improvement to above-mentioned technical proposal, end amino acid L is by amination.

As further improvement to above-mentioned technical proposal, the polypeptide toxin includes SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

Another object of the present invention is to provide a kind of fusion protein, the fusion protein includes aforementioned polypeptides toxin.

Another object of the present invention is to provide a kind of polynucleotides, the polynucleotide encoding aforementioned polypeptides toxin.

Another object of the present invention is to provide a kind of carrier, the carrier includes the multicore glycosides of encoding such polypeptides toxin Acid.

Another object of the present invention is to provide a kind of host cell, the host cell includes above-mentioned carrier.

Another object of the present invention is to provide a kind of pharmaceutical composition, described pharmaceutical composition aforementioned polypeptides toxin and medicine Acceptable excipient on.

Another object of the present invention is to provide aforementioned polypeptides toxin in preparation for treating the relevant disease of Nav1.7 Purposes in drug.

As further improvement to above-mentioned technical proposal, wherein the relevant disease of the Nav1.7 is pain.

As further improvement to above-mentioned technical proposal, wherein pain is chronic ache, Acute Pain, nerve pain Bitterly, nociceptive pain, visceral pain, back pain, postoperative pain, hot pain, phantom limb pain or pain associated with inflammatory conditions Bitterly, Primary Erythermalgia (PE), paroxysmal severe pain disease (PEPD), osteoarthritis, rheumatoid arthritis, lumbar intervertebral disc cutting It removes, neuropathy (PHN), trigeminal neuralgia after pancreatitis, fibromyalgia, pain diabetic neuropathy (PDN), bleb (TN), spinal cord injury or multiple sclerosis.

The existing sodium ion blocking agent derived from natural toxin does not have selectivity, while blocking Nav1.7 eases pain Nav1.4 can be blocked, 1.5,1.6 equal sodium-ion channels can interfere breathing and heartbeat, will lead to breath interruption when serious, heartbeat stops Etc. lethal effects, so cannot act as analgesic.Polypeptide toxin of the invention is effective specific inhibitor of Nav1.7, with Existing toxin is compared, and polypeptide toxin of the invention can enhance selectivity, can only selectivity inhibition Nav1.7 without Inhibit other sodium-ion channels, to avoid above-mentioned side effect, thus can be used as analgesic.

Specific embodiment

Unless otherwise defined, the meaning of all technical and scientific terms used herein with it is of the art The meaning that those of ordinary skill is generally understood is identical.Although this document describes illustrative composition and method, it is similar or Any composition and method for being equal to composition as described herein and method can be used in practice or test of the invention.

Term as used herein " polypeptide " refers to the change being made up of two or more amino acid Covalent bonding together Object is closed, the covalent bond is by eliminating H from the carboxyl of the amino group of amino acid and next amino acid₂O molecule And formed.

Term " polynucleotides " refers to comprising being covalently attached by sugar-phosphate backbone or other equivalent covalent chemical modes Nucleotide chain molecule.Double-stranded DNA and single stranded DNA and double-stranded RNA and single stranded RNA are the typical cases of polynucleotides.This The polynucleotides of invention can also include at least one non-coding sequence, the sequence such as transcribed but do not translated, termination signal, core Sugared body binding site, mRNA critical sequences, introne and polyadenylation signal.Polynucleotide sequence can also be another comprising coding The other sequence of outer amino acid.These other polynucleotide sequences can (for example) coded markings or well known label sequence Column, such as hexahistine or HA label, these labels are conducive to the purifying of fused polypeptide.

Term " carrier " is the non-natural multicore for referring to replicate or can move between such system in biosystem Thuja acid.Vector polynucleotides generally comprise the cDNA and other element of encoding target protein, such as replication orgin, poly- gland Nucleotide signal or selected marker, function are to promote the duplication or holding of these polynucleotides in biosystem.Such life The example of object system may include cell, virus, animal, plant and the biology department reconstructed with the biological components for capableing of replicating vector System.Polynucleotides comprising carrier can be DNA or the hybrid molecule of RNA molecule or these molecules.

Term " host cell " refers to the cell for having been introduced into carrier.It should be understood that term host cell is not only intended to refer to spy Fixed host cell also refers to the filial generation of this cell.Because certain can occurs in offspring due to mutation or since environment is influenced A little modifications, therefore this filial generation can be different from mother cell, but are included in the range of the term as used herein " host cell " It is interior.Such host cell can be eukaryocyte, prokaryotic cell, plant cell or archaeal cell.

" pharmaceutical composition " includes polypeptide toxin and pharmaceutically acceptable excipient of the invention.Suitable medium or Carrier can be water for injection, normal saline solution or artificial cerebrospinal fluid, may be supplemented in the composition of parenterai administration Common other materials.Other exemplary media object is neutral buffered saline or is mixed with sero-abluminous salt water.These Solution is sterile, and substantially free of particulate matter, and can be carried out by the sterilization technology (for example, filtering) of conventional well known Sterilization.Composition may include close to pharmaceutically acceptable excipient needed for physiological condition, such as pH adjusting agent and buffer, Stabilizer, thickener, lubricant and coloring agent etc..This hair according to selected specific administration mode, in such pharmaceutical composition Bright polypeptide toxin concentration can widely change, for example, about 0.1 weight %, 0.2 weight %, 0.3 weight %, 0.4 weight %, 0.5 weight %, 0.6 weight %, 0.7 weight %, 0.8 weight %, 0.9 weight %, 1.0 weight %, 1.1 weight %, 1.2 weights Measure %, 1.3 weight %, 1.4 weight %, 1.5 weight %, 1.6 weight %, 1.7 weight %, 1.8 weight %, 1.9 weight %, 2 Weight %, or in 2 weight % between 5 weight %, up to 15 weight %, 20 weight %, 30 weight %, 40 weight %, 50 Weight %, 60 weight % or 70 weight %, and be based primarily upon fluid volume, viscosity and other factors and selected.

In entire the application, the description of each embodiment uses the language of " comprising "；However, those skilled in the art will It is understood that in certain specific examples, can alternatively using language "consisting essentially of" or " by ... form " Lai Embodiment is described.

Polypeptide toxin described herein can be used for treating pain or inducing analgesia.As it is used herein, term " is controlled Treat " it also include the pain that prevention has the patient or subject of developing such pain tendency, and change when pain is determined Peculiar symptom that is kind or eliminating the pain developed or the such pain of mitigation.

Pain as described herein can be the pain of any known type, including periphery property pain.For example, pain to be treated Pain can be one or more pain, be described as neuropathic pain, neuropathic pain, cancer pain, postoperative pain, mouth Chamber or dental pain, the pain from mentioned trigeminal neuralgia, the pain from postherpetic neuralgia or since reflection is handed over Pain and/or pain relevant to inflammatory conditions caused by perceptual malnutrition.

Pain can be characterized as example, nociceptive pain, non-nociceptive pain, somatalgia, splanchnodynia, neuralgia.Pain Can be hot stimulation responses, cold, vibration, the chemical irritant for stretching and being discharged by damaged cell receptor result.This Outside, pain can be the result of the pain as caused by nerve cell dysfunction.Theme according to the present invention, pain can be Pain-relevant to tissue such as skin, muscle, joint, bone and ligament is commonly referred to as musculoskeletal pain.Pain is also possible to Pain relevant to the internal organs of main cavity.In this respect, pain can be with chest (heart and lung), abdomen (liver,kidney,spleen And intestines) and/or pelvic cavity (bladder, uterus and ovary) it is related.In addition, pain described according to the inventive subject matter can for The relevant pain of nervous system itself, such as pain related with pinching nerve or pressuring nerve.Pain may originate from peripheral nerve Nerve between system, i.e. tissue and spinal cord, or it is derived from central nervous system, i.e. nerve between spinal cord and brain.Pain can With for example by multiple sclerosis, apoplexy, cerebral hemorrhage and/or anoxic, compression of nerves, pressuring nerve, neuroinflamation, interverbebral disc it is damaged Or slippage and/or the nerve infection nerve degeneration as caused by shingles zoster or other virus infections are related.

Inflammatory conditions can be related to one or more illnesss, and the illness is selected from by Acute Pain, migraine, headache, Migraine, traumatic nerve damage, neurothlipsis, Nerve entrapments, postherpetic neuralgia, trigeminal neuralgia, diabetes Nerve lesion, chronic low-back pain, phantom limb pain, chronic pelvic pain, neuroma pain, complex region Pain Syndrome are chronic Arthritis ache, and with the neuropathy of cancer, chemotherapy, HIV and HIV therapy induction, intestinal irritable syndrome and have related disorders The group of pain composition relevant with Crohn disease.

It is that " treatment " or " processing " of term disease, obstruction and illness includes mitigating its at least one as used herein Symptom reduces its severity, or delays, and prevents or inhibits its progress.Treatment does not need to refer to that disease, obstruction and illness are controlled completely More.In order to effectively treat, useful composition as described herein only needs to reduce disease, the severity of obstruction and illness, reduces The severity of relative symptom, improves the quality of life of patient or subject, or delays, and prevents or inhibits disease, barrier Hinder or the breaking-out of illness.

As used herein other terms are defined by meaning known in the art.

Synthesis polypeptide toxin

Recombinant DNA technology can be prepared or can be used by chemical synthesis produce polypeptide toxin described herein.For Polypeptide toxin of the invention is prepared by chemical synthesis, disclosed known method can be used, such as can be by using folded Nitride, acid chloride, acid anhydrides, compound acid anhydrides, DCC, Acibenzolar, woodward's reagent K, carbonylic imidazole, deoxidation, DCC/ HONB, bop reagent method obtain polypeptide toxin of the invention.In addition, automatic peptide synthesizer (such as PE can be used Applied Bio Systems Co.) peptide described herein is prepared by chemical synthesis.Such as in Bulaj G. et al. (2006) 45 Biochemistry, method described in 7404 can also be used for synthesizing peptide described herein and refolding program In.

In addition, after completion of the reaction, can by published known purification methods to polypeptide toxin described herein into Row purifying and separation.For example, can be by the combination of solvent extraction, distillation, column chromatography, liquid chromatogram, recrystallization etc. to the present invention Polypeptide toxin purified and separated.When the polypeptide toxin as described herein obtained by the above method is free form, Disclosed known method can be used to be converted into salt form, and on the other hand, when the polypeptide toxin of acquisition is salt form, Disclosed known method can be used to be converted into free form.

Embodiment 1

The amino acid sequence of the present embodiment GPTX-1 polypeptide toxin is as shown in table 1 below.

Table 1

Remarks: the L amino acid of the end SEQ ID NO:5~SEQ ID NO:8 has carried out amination, that is, added amino. Experimental method

1, pharmaceutical samples

Intracellular fluid: 145mM KCl, 5mM NaCl, 2mM CaCl2,2mM MgCl2,4mM ethylene glycol tetraacetic (ethylene glycol tetra-acetic acid),10mM HEPES；KOH pH 7.3；Osmotic pressure: 300m Osmol； Extracellular fluid: 140mM NaCl, 4mM KCl, 1mM MgCl2,1.8mM CaCl2,10mM glucose monohydrate (D-glucose monohydrate,)10mM HEPES；HCl pH 7.4；

Glass electrode: WPI:1B150-3；

Cell: HEK293 cell (commercialization purchase) expresses NaV electric current.

2, key instrument

Electrode draws instrument (U.S. Sutter；P97)；

Patch Clamp System (German HEKA；EPC-10)；

Osmotic pressure analyzer (U.S. Wescor；)；

Micro-sampling pin (U.S. Hamilton；)；

PH analyzer (Italian Mick Instrument Ltd.).

3, experimental procedure

3.1 cell culture

The recovery of cell

(1) equilibrium at room temperature culture medium takes out the culture dish/bottle for being coated with matrix (Matrix), sucks coating buffer and be added Appropriate culture medium, is placed in 5%CO₂37 DEG C of constant temperature cell incubators in；

(2) 37 DEG C of defrosting cells, quickly rocking, which melts cell to an only surplus fritter ice crystal, (avoids cryopreservation tube lid from contacting water Face), it takes out rapidly and freezes tube outer surface with 75% alcohol disinfecting；

(3) with 1mL pipettor it is careful cell suspension is transferred to a new 15mL centrifuge tube, avoid violent pressure-vaccum；

(4) cryopreservation tube is cleaned with 1mL culture medium, to collect remaining cell, be then added dropwise containing cell suspension 15mL centrifuge tube should maintain 5 seconds 1 slow rates for dripping (50 μ L), 1 minute left side since the dilution ratio being initially added dropwise is larger The right side drips this 1mL culture medium, and constantly jog mixes during being added dropwise；

(5) with the rate of 1 drop per second, 4mL culture medium is added dropwise, constantly jog mixes during being added dropwise；

(6) 200g is centrifuged 5min；

(7) cell is resuspended with culture medium, soft piping and druming mixes, and is inoculated into ready culture dish/bottle, planche cross It shakes up；

(8) 5%CO₂37 DEG C of constant incubators in cultivate 48h, replace culture medium.

Maintain culture

(1) equilibrium at room temperature culture medium；

(2) the cell culture medium culture obtained, every 48h are changed the liquid once；Or the maintenance culture of standard volume 150% is added Base, every 72h are changed the liquid once.

3.2 patch-clamps detect electric current

(1) extracellular fluid and intracellular fluid are prepared in advance:

(2) it using perfusion system to perfusion extracellular fluid in incubation slot, flow velocity 5ml/min, and adjusts warm in incubation slot Degree is 35-36 DEG C.

(3) whole-cell recording technique: cultured cell is taken out from incubator, culture solution is discarded, extracellular fluid is added, place In the case where just setting microscope, chooses smooth cell and recorded；

(4) glass electrode is drawn, the resistance after electrode nightfall is drawn using corresponding program is 3-5M Ω；

(5) so that recording electrode is just pressed into water by Micromanipulators, persistently adjust micro- behaviour, make recording electrode exposing cell table Face, at this time due to the unexpected increase of electrode resistance, the decline of current value representated by the sealing-in test pulse that film test window is shown, Positive pressure is removed, and imposes 10~30cm H₂O negative pressure, observation sealing-in resistance rises rapidly, until reaching Giga-Ohm seal.

(6) electrode capacitance compensation is carried out, after offsetting the temporary charge reservoir electric current generated due to recording electrode, continues to increase negative pressure To rupture of membranes, whole-cell recording technique is formed.

(7) it observes the capacitance current from entire cell membrane, carries out membrane capacitance compensation and series resistance compensation, offset institute Caused by voltage drop, make as far as possible observation current line it is smooth

(8) mother liquor that drug is configured to 2000X is stored for future use in -20 DEG C, takes the electrode external solution of 50mL according to corresponding end Concentration is configured to work medical fluid.Perfusion work medical fluid 5min, then uses normal cell external solution after the stable record 5min of system By the medical fluid elution record 5min in chamber.

(9) suppression percentage and IC50 numerical value are calculated.

Experimental result is as shown in table 2 below.

Table 2

Remarks: N.D. is indicated beyond monitoring limiting value, without data

Sequence table

<110>Qinghai Fen Tuolihua Biotechnology Co., Ltd

<120>Ccotx1 toxin and its application

<130>

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 33

<212> PRT

<213>artificial sequence

<400> 1

Asp Cys Leu Gly Trp Phe Lys Ser Cys Asp Pro Lys Asn Asp Lys

1 5 10 15

Cys Cys Lys Asn Tyr Thr Cys Ser Arg Arg Asp Arg Trp Cys Lys

20 25 30

Tyr Asp Leu

<210> 2

<211> 33

<212> PRT

<213>artificial sequence

<220>

<223>peptide

<220>

<221>other features

<222>(1) ... (1)

<223>Xaa1 is independently selected from Asp, Ile and Glx

<220>

<221>other features

<222>(5) ... (5)

<223>Xaa5 is independently selected from Ala, Ile and Met

<220>

<221>other features

<222>(7) ... (7)

<223>Xaa7 is independently selected from Lys and Ala

<220>

<221>other features

<222>(8) ... (8)

<223>Xaa8 is independently selected from Ser

<220>

<221>other features

<222>(10) ... (10)

<223>Xaa10 is independently selected from Asp

<220>

<221>other features

<222>(12) ... (12)

<223>Xaa12 is independently selected from Glu and Lys

<220>

<221>other features

<222>(18) ... (18)

<223>Xaa18 is independently selected from Tyr and Lys

<220>

<221>other features

<222>(19) ... (19)

<223>Xaa19 is independently selected from Arg and Asn

<220>

<221>other features

<222>(20) ... (20)

<223>Xaa20 is independently selected from Leu and Tyr

<220>

<221>other features

<222>(21) ... (21)

<223>Xaa21 is independently selected from Val and Thr

<220>

<221>other features

<222>(24) ... (24)

<223>Xaa24 is independently selected from Lys and Arg

<220>

<221>other features

<222>(25) ... (25)

<223>Xaa25 is independently selected from Ser and Arg

<220>

<221>other features

<222>(26) ... (26)

<223>Xaa26 is independently selected from His and Asp

<220>

<221>other features

<222>(27) ... (27)

<223>Xaa27 is independently selected from Asn and Arg

<220>

<221>other features

<222>(31) ... (31)

<223>Xaa31 is independently selected from Trp and Tyr

<220>

<221>other features

<222>(32) ... (32)

<223>Xaa32 is independently selected from Lys and Asp

<400> 2

Xaa Cys Leu Gly Xaa Phe Xaa Xaa Cys Xaa Pro Xaa Asn Asp Lys

1 5 10 15

Cys Cys Xaa Xaa Xaa Xaa Cys Ser Xaa Xaa Xaa Xaa Trp Cys Lys

20 25 30

Xaa Xaa Leu

<210> 3

<211> 33

<212> PRT

<213>artificial sequence

<400> 3

Asp Cys Leu Gly Met Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 4

<211> 33

<212> PRT

<213>artificial sequence

<400> 4

Ile Cys Leu Gly Met Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 5

<211> 33

<212> PRT

<213>artificial sequence

<400> 5

Ile Cys Leu Gly Met Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 6

<211> 33

<212> PRT

<213>artificial sequence

<400> 6

Glx Cys Leu Gly Ile Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 7

<211> 33

<212> PRT

<213>artificial sequence

<400> 7

Glx Cys Leu Gly Ile Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Tyr Arg Leu Val Cys Ser Lys Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 8

<211> 33

<212> PRT

<213>artificial sequence

<400> 8

Glx Cys Leu Gly Ile Phe Lys Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Asn Trp Cys Lys

20 25 30

Trp Lys Leu

<210> 9

<211> 33

<212> PRT

<213>artificial sequence

<400>

Asp Cys Leu Gly Ala Phe Ala Ser Cys Asp Pro Glu Asn Asp Lys

1 5 10 15

Cys Cys Lys Arg Leu Val Cys Ser Arg Ser His Arg Trp Cys Lys

20 25 30

Trp Lys Leu

Claims (11)

1.Ccotx1 toxin, the polypeptide toxin include following SEQ ID NO:2:

X₁CLGX₅FX₇X₈CX₁₀PX₁₂NDKCCX₁₈X₁₉X₂₀X₂₁CSX₂₄X₂₅X₂₆X₂₇WCKX₃₁X₃₂L, wherein

X₁For D, I or Z；

X₅For A, I or M；

X₇For K or A；

X₈For S；

X₁₀For D；

X₁₂For E or K；

X₁₈For Y or K；

X₁₉For R or N；

X₂₀For L or Y；

X₂₁For V or T；

X₂₄For K or R；

X₂₅For S or R；

X₂₆For H or D；

X₂₇For N or R；

X₃₁For W or Y；

X₃₂For K or D.

2. polypeptide toxin as described in claim 1, end amino acid L is by amination.

3. polypeptide toxin as claimed in claim 2, the polypeptide toxin includes SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

4. a kind of fusion protein, the fusion protein includes polypeptide toxin as claimed in any one of claims 1 to 3.

5. a kind of polynucleotides, the polynucleotide encoding polypeptide toxin as claimed in any one of claims 1 to 3.

6. a kind of carrier, the carrier includes polynucleotides as claimed in claim 5.

7. a kind of host cell, the host cell includes carrier as claimed in claim 6.

8. a kind of pharmaceutical composition, described pharmaceutical composition includes polypeptide toxin as claimed in any one of claims 1 to 3 and medicine Acceptable excipient on.

9. polypeptide toxin as claimed in any one of claims 1 to 3 is preparing the drug for treating the relevant disease of Nav1.7 In purposes.

10. purposes according to claim 9, wherein the relevant disease of the Nav1.7 is pain.

11. purposes according to claim 10, wherein pain is chronic ache, Acute Pain, neuropathic pain, nocuity Pain, visceral pain, back pain, postoperative pain, hot pain, phantom limb pain or pain associated with inflammatory conditions, primary Acromelalgia (PE), paroxysmal severe pain disease (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreas Neuropathy (PHN), trigeminal neuralgia (TN), spinal cord damage after inflammation, fibromyalgia, pain diabetic neuropathy (PDN), bleb Wound or multiple sclerosis.