CN101792485B - Alpha-type conus polypeptide and application thereof - Google Patents

Abstract

The invention discloses alpha-type conus polypeptides and application thereof. The invention provides seven polypeptides: in the polypeptide I, an amino acid sequence is disclosed by a sequence 7 in a sequence table, and the last amino acid residue is amidated; in the polypeptide II, an amino acid sequence is disclosed by a sequence 8 in the sequence table, and the last amino acid residue is amidated; in the polypeptide III, an amino acid sequence is disclosed by a sequence 9 in the sequence table, and the last amino acid residue is amidated; in the polypeptide IV, an amino acid sequence is disclosed by a sequence 10 in the sequence table, and the last amino acid residue is amidated; in the polypeptide V, an amino acid sequence is disclosed by a sequence 11 in the sequence table, and the last amino acid residue is amidated; in the polypeptide VI, an amino acid sequence is disclosed by a sequence 12 in the sequence table, and the last amino acid residue is amidated; and in the polypeptide VII, an amino acid sequence is disclosed by a sequence 13 in the sequence table. By the determination of activity, the seven polypeptides of a general formula I have abirritation, wherein the alpha-type conus polypeptides T-1, T-2, T-3 and T-4 have obvious analgesic activity, T5-T7 also have obvious analgesic activity, and the alpha-type conus polypeptides have application value in an analgesic aspect.

Description

Alpha-type conus polypeptide and application thereof

Technical field

The present invention relates to a kind of alpha-type conus polypeptide and application thereof.

Background technology

Kind surplus Conus gasteropod, the whole world have 500 approximately spreads all over each warm sea area, the world, and there is cone shell kind more than 100 in China, mainly is distributed in the Xisha Islands, Hainan Island and the marine site, Taiwan of China.Conotoxin peptide is secreted by the poison gland of cone shell venom duct and malicious capsule inner wall, contains 50-200 active polypeptide in the venom of every kind of cone shell, and the contained bioactive peptide of different varieties cone shell has nothing in common with each other; Even cone shell of the same race is different because of the marine site, its toxin composition also can there are differences, and estimates to exist approximately the polypeptide (McIntosh of more than 50,000 kind of different activities in theory; J.M., Jones, R.M. (2001) .Conevenom-from accidental stings to deliberate injection.Toxicon; 39,1447-1451.Nelson, L. (2004) .Venomous snails:One slip; And you ' re dead.Nature, 429,798-799.).Conotoxin peptide ability specific effect is in the various receptor subtypes of neurotransmitters such as vagusstoff, and different kinds of ions passages such as calcium, sodium and potassium, not only can also be used for the lead compound of developing new drug as the ideal molecular model directly as medicine.Wherein, ω type conotoxin peptide MVIIA is come into the market by the medicine of FDA approval as treatment HIV and TCA pain; The SO-3 that from South China Sea strain line cone shell, finds also has very strong analgesic activities (Daiqy et al.J Nat Prod 2003,66 (9) 1,276-9; Patent Zl 00128525.4).But above-mentioned two kinds of conotoxin peptides act on the N-type calcium channel of nervus centralis, and administering mode is in the sheath, is inconvenient to use.

(α-CTX) belong to conotoxin peptide A superfamily (being divided into A, M, O, P, I, J, S, superfamilies such as T by signal peptide conserved sequence and disulfide linkage pattern of rows and columns conotoxin peptide), specific action is in muscularity and neuron pattern nAChRs for α-conotoxin peptide.Most α-conotoxin peptides contain 12-18 amino acid, two couples of disulfide linkage (CCX _mCX _nC, mode of connection is 1-3,2-4).According to m, the number of n is different, and α-conotoxin peptide is subdivided into α 3/5, and α 4/3, and α 4/4, and α 4/6, α 4/7 subfamily.Find that at present some α-conotoxin peptide has analgesic activities, wherein α-conotoxin peptide Vc1.1 (GCCSDPRCNYDHPEICONH ₂) be from Victoria cone shell (Conus Victoriae), form by 16 amino acid, contain two pairs of disulfide linkage (arrangement mode of disulfide linkage is 1-3,2-4) (Sandall et al.Biochemistry2003,42,6904-6911.).Research shows that Vc1.1 has shown preferably analgesic activities and had in the neuropathic pain animal model and quickens effect (the Satkunanathan et al.Brain Res 2005 that injured nerve recovers; 1059; 149-158), got into the clinical study stage in 2006.Another kind of alpha-type conus polypeptide RgIa shows analgesic activity equally in animal model.This shows; Exist some to have the polypeptide of better analgesic activities in the alpha-type conus polypeptide; They are significant for the medicine of Development of New Generation neuropathic pain, and these polypeptide can adopt administering modes such as subcutaneous, muscle or abdominal cavity, greatly facilitate application.

Different according to the cause of disease of nerve injury, nature and extent, be divided into the peripheral nerve pain two big classes due to nervus centralis pain and the peripheral nerve injury clinically.Wherein, nervus centralis pain is called for short central pain, is the primary pain that the generation infringement of pain conduction path or the dysfunction of cns causes, is common in wound, cerebrovascular disease or the multiple sclerosis and the tumour etc. of spinal cord.Peripheral nerve pain is that factors such as wound, ischemic, compressing, infection, inflammation or metabolism are damaged due to the peripheral nerves, like phantom limb pain, PHN, polyneuritis, diabetic peripheral neuralgia etc.The classical symptom of neuropathic pain comprises paresthesia, hyperpathia and allodynia etc.Medicine commonly used clinically mainly contains the coupling of anticonvulsive drug (carrying western phthalein, oxcarbazepine like Lay), opiates medicine (like morphine) and various thymoleptic; But because problems such as the tolerance that toxic side effect and long-term prescription bring, habituation property; Not good (the Dray et al of result of treatment; Br J Anaesth.2008,101 (1), 48-58; Gilron et al, Expert OpinEmerg Drugs.2007,12 (1), 113-26.).Therefore, find analgesic ten minutes necessity of high reactivity of new generation, low tolerance and non-habituation.

Summary of the invention

The purpose of this invention is to provide a kind of alpha-type conus polypeptide and application thereof.

Alpha-type conus polypeptide provided by the invention is the polypeptide shown in the general formula I;

X ₁-X ₂CCX ₃-X ₄-X ₅-X ₆CX ₇-X ₈-X ₉-X ₁₀-X ₁₁-X ₁₂-X ₁₃-X ₁₄C-β

General formula I

In the general formula I:

X ₁Be selected from any amino acid or vacancy among G, the R;

X ₂Be selected from any amino acid among G, N, the K;

X ₃Be selected from any amino acid among S, T, D, the M;

X ₄Be selected from any amino acid among N, R, I, the F;

X ₅Be selected from any amino acid among P, S, the H;

X ₆Be selected from any amino acid among A, F, D, the T;

X ₇Be selected from any amino acid among M, K, Y, P, the N;

X ₈Be selected from any amino acid among L, R, N, the I;

X ₉Be selected from any amino acid among K, I, D, the Y;

X ₁₀Be selected from any amino acid among Y, the N;

X ₁₁Be selected from any amino acid among P, R, S, the K;

X ₁₂Be selected from any amino acid among N, D, E, R, the L;

X ₁₃Be selected from any amino acid or vacancy among L, Q, the F;

X ₁₄Be selected from any amino acid or vacancy among N, the Y;

β is selected from carboxamido-group or carboxyl;

Above-mentioned letter definition is following: the D-aspartic acid; E-L-glutamic acid; The I-Isoleucine; K-Methionin; The L-leucine; The M-methionine(Met); The N-l-asparagine; The Q-Stimulina; The R-l-arginine; The S-Serine; The T-Threonine; The G-glycocoll; The F-phenylalanine(Phe); The P-proline(Pro); The H-Histidine; The A-L-Ala; Y-tyrosine.

Said polypeptide specifically can be any one in following seven peptide species:

Polypeptide I: aminoacid sequence is shown in the sequence 7 of sequence table, and last amino acids residue amidation;

Polypeptide II: aminoacid sequence is shown in the sequence 8 of sequence table, and last amino acids residue amidation;

Polypeptide III: aminoacid sequence is shown in the sequence 9 of sequence table, and last amino acids residue amidation;

Polypeptide IV: aminoacid sequence is shown in the sequence 10 of sequence table, and last amino acids residue amidation;

Polypeptide V: aminoacid sequence is shown in the sequence 11 of sequence table, and last amino acids residue amidation;

Polypeptide VI: aminoacid sequence is shown in the sequence 12 of sequence table, and last amino acids residue amidation;

Polypeptide VII: aminoacid sequence is shown in the sequence 13 of sequence table.

7 amino acid sequence of polypeptide are following:

Peptide T 1:NH ₂-GCCSNPACMLKNPNLC-CONH ₂

Peptide T 2:NH ₂-NCCTRSFCKRIYPDLC-CONH ₂

Peptide T 3:NH ₂-GCCDIPDCYNKNREQC-CONH ₂

Peptide T 4:NH ₂-NCCMFHTCPIDYSRFNC-CONH ₂

Peptide T 5:NH ₂-GNCCMFHTCPIDYSRFNC-CONH ₂

Peptide T 6:NH ₂-GNCCMFHTCPIDYSRFYC-CONH ₂

Peptide T 7:NH ₂-RKCCSNPACNRYNKLC-COOH.

The preparation method of said polypeptide II is specific as follows: the chemosynthesis aminoacid sequence is dissolved in the NH of 0.1M shown in the sequence 8 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.2, and magnetic agitation 24-48h obtains polypeptide II.The preparation method of said polypeptide III is specific as follows: the chemosynthesis aminoacid sequence is dissolved in the NH of 0.1M shown in the sequence 9 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.2, and magnetic agitation 24-48h obtains polypeptide III.The preparation method of said polypeptide IV is specific as follows: the chemosynthesis aminoacid sequence is dissolved in the NH of 0.1M shown in the sequence 10 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.2, and magnetic agitation 24-48h obtains polypeptide IV.The preparation method of said polypeptide V is specific as follows: the chemosynthesis aminoacid sequence is dissolved in the NH of 0.1M shown in the sequence 11 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.2, and magnetic agitation 24-48h obtains polypeptide V.The preparation method of said polypeptide VI is specific as follows: the chemosynthesis aminoacid sequence is dissolved in the NH of 0.1M shown in the sequence 12 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.2, and magnetic agitation 24-48h obtains polypeptide VI.The preparation method of said polypeptide I is specific as follows: the chemosynthesis aminoacid sequence is shown in the sequence 7 of sequence table and the amidated linear polypeptide of last amino acids residue; Place pH7.7 0.1M Tris-HCl damping fluid oxidation 28h; Add the acetic acid termination reaction then; The desalination freeze-drying with 10mM iodine solution lucifuge reaction 10min, obtains polypeptide I then.The preparation method of said polypeptide VII is specific as follows: the linear polypeptide shown in the sequence 13 of chemosynthesis sequence table; Place pH7.7 0.1M Tris-HCl damping fluid oxidation 28h; Add the acetic acid termination reaction then; The desalination freeze-drying with 10mM iodine solution lucifuge reaction 10min, obtains polypeptide VII then.

The encoding sox of said polypeptide also belongs to protection scope of the present invention.

The encoding sox of said polypeptide specifically can be any one in following seven kinds of dna fragmentations:

(1) sequence 1 of sequence table is from the dna molecular shown in the 142nd to 189 Nucleotide of 5 ' end;

(2) sequence 2 of sequence table is from the dna molecular shown in the 142nd to 189 Nucleotide of 5 ' end;

(3) sequence 3 of sequence table is from the dna molecular shown in the 133rd to 180 Nucleotide of 5 ' end;

(4) sequence 4 of sequence table is from the dna molecular shown in the 139th to 189 Nucleotide of 5 ' end;

(5) sequence 5 of sequence table is from the dna molecular shown in the 136th to 189 Nucleotide of 5 ' end;

(6) sequence 6 of sequence table is from the dna molecular shown in the 136th to 189 Nucleotide of 5 ' end;

(7) sequence 7 of sequence table is from the dna molecular shown in the 133rd to 180 Nucleotide of 5 ' end.

The present invention also protects a kind of analgesic medicine that is used for, its activeconstituents be more than arbitrary described polypeptide.

More than arbitrary described polypeptide also belong to protection scope of the present invention in the application that preparation is used for the analgesic medicine.

The present invention also protects a kind of analgesic agent, is above arbitrary described polypeptide.

More than arbitrary described polypeptide or all can be applicable to prepare analgesic agent.

Above-mentioned alpha-type conus polypeptide can adopt Fmoc method solid phase synthesis, uses the high-efficient liquid phase chromatogram technology purifying.

7 polypeptide finding general formula I through determination of activity have analgesic activity, and wherein α conotoxin peptide T-1, T-2, T-3, T-4 have significant analgesic activities, and T5-T7 also has obvious analgesic activities, have using value at the inhibition ease pain.

The verivate of described alpha-type conus polypeptide also belongs to protection scope of the present invention.The verivate of described alpha-type conus polypeptide can be as follows (I), (II), (III) or (IV):

(I) said alpha-type conus polypeptide is carried out one or more amino acid whose insertions and/or replacement and/or disappearance, the verivate of the alpha-type conus polypeptide that obtains;

(II) with said alpha-type conus polypeptide or (I) verivate of alpha-type conus polypeptide carry out cyclisation, fatization or PEGization modification, the verivate of the alpha-type conus polypeptide that obtains;

(III) with said alpha-type conus polypeptide or (I) verivate of alpha-type conus polypeptide connect the verivate of the alpha-type conus polypeptide that obtains with carrier;

(IV) with one or several said alpha-type conus polypeptide or (I) verivate of alpha-type conus polypeptide be connected to polymer, the verivate of the alpha-type conus polypeptide that obtains.

In the described alpha-type conus polypeptide, can modify side chain amino acid, the modification of these residues can strengthen the biological activity of peptide or the selectivity of target.

Said replacement can also be " conservative property replacement " or " non-conservation replacement ".Replacement can be an amino acid whose replacement, also can be a plurality of amino acid whose replacements, can be that successive replaces, and also can be that dispersive replaces." conservative property replacement ": use conformation as the amino acid of D-type, the rare amino acid of nature existence or manually modified amino acid replacement, to increase bioavailability and to improve and suppress activity the one or more amino-acid residues in the described alpha-type conus polypeptide; The amino acid of D-type refers to the relative amino acid of amino acid with the L-type of constitutive protein matter; The rare amino acid that nature exists comprises the not common amino acid of constitutive protein matter and the amino acid of constitutive protein matter not, like 5-oxylysine, methylhistidine, γ-An Jidingsuan, homoserine etc.; Manually modified amino acid refers to through methylating the common L-type amino acid of the constitutive protein matter that phosphorylation etc. are modified." non-conservation replacement ": an amino acid or a plurality of amino-acid residue in the polypeptide are replaced by amino acid of different nature, are perhaps replaced by unconventional amino acid.

Said interpolation can be an amino acid or a plurality of natural or unconventional amino acid.

Said disappearance also comprises one or more amino acid whose disappearance.

Said carrier includes, but are not limited to protein, polyoxyethylene glycol, lipid material etc.

Several (1-4) identical alpha-type conus polypeptide can pass through amino acid, and like Methionin, halfcystine, perhaps other molecule is joined together to form polymer.

Also can contain in opiate receptor antagonist, opiate receptor partial agonist, norepinephrine receptor antagonist, α 2 norepinephrine receptor agonists, cholinergic nerve antagonist, calcium channel blocker or the nmda receptor antagonist any one or a few in the said medicine, to strengthen its analgesic effect.

The pharmaceutics acceptable auxiliary be can add in the said medicine, injection, oral preparation, rectum body processed to different dosage forms such as medicament or skin-absorbent agents.

Alpha-type conus polypeptide of the present invention has following characteristics:

1, alpha-type conus polypeptide of the present invention has very strong analgesic activities; Wherein T-2, T-3, four peptides of T-4 both can make threshold of pain rate improve more than 40% at intramuscular injection dosage (10nmol/kg) under very low situation; The threshold of pain rate that can make T-1 improves more than 80%, and T-5, T-6, T-7 also can make the threshold of pain improve more than 20%.

2, alpha-type conus polypeptide of the present invention is from the South China Sea cone shell, has brand-new amino acid and forms, and on amino acid is formed, has significant difference with the alpha-type conus polypeptide of present report.

3, alpha-type conus polypeptide of the present invention acts on peripheral nervous system, and administering mode is subcutaneous, muscle or abdominal injection, compares with the intrathecal drug delivery mode of existing analgesic MVIIA, and it is more convenient to use.

Description of drawings

Fig. 1 is the stratographic analysis figure behind the folding 24h of T-2, T-3, T-4, T-5, T-6 and Vc1.1; The folding figure of A:T-2; The folding figure of B:T-3; The folding figure of C:T-4; The folding figure of D:T-5; The folding figure of E:T-6; The folding figure of F:Vc1.1.

Fig. 2 is the stratographic analysis figure of folding and second step of T-1, the T-7 the first step after folding; The folding figure of A:T-1 the first step oxidation; The folding figure of the B:T-1 second step oxidation; The folding figure of C:T-7 the first step oxidation; The folding figure of the D:T-7 second step oxidation.

Fig. 3 is the stratographic analysis figure behind T-1, T-2, T-3, T-4, T-5, T-6, T-7, the Vc1.1 peptide purification; A:T-1 purity analysis figure; B:T-2 purity analysis figure; C:T-3 purity analysis figure; D:T-4 purity analysis figure; E:T-5 purity analysis figure; F:T-6 purity analysis figure; G:T-7 purity analysis figure; H:Vc1.1 purity analysis figure.

Fig. 4 is the analgesic activities of T-1, T-2, T-3, T-4, T-5, T-6, T-7, Vc1.1 polypeptide.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

The discovery of embodiment 1, alpha-type conus polypeptide

1, the extraction of the total RNA of cone shell

From the Xisha Islands, ground such as Sanya, Hainan gathers 6 kinds of cone shell live bodies as sample material, adopts the Trizol method to extract total RNA of 6 kinds of cone shells respectively.6 kinds of cone shells: marble cone shell (Conus marmoreus), grand cone shell (Conusimperialis), conus (Conus litteratus), evil mind cone shell (Conus emaciatus), maiden cone shell (Conus eberneus), calf cone shell (Conus vitulinus).

The Trizol method is extracted the concrete steps of total RNA:

1. prior to getting cone shell poison gland pipe on ice, be placed in the liquid nitrogen freezingly and use the mill grinding powder, pour in the micro-homogenizer of prior ice bath.

2. drawing 1.2ml Trizol adding homogenizer and further grinding makes cone shell organize cracking to discharge RNA.

3. ground slurries are poured in the centrifuge tube of 1.5ml, room temperature leaves standstill 5min.

4. add 20% chloroform, 240 μ l, behind the concuss 15s, be put in 10min on ice.

5. 4 ℃, the centrifugal 15min of 12000rpm carefully shift in the new centrifuge tube of supernatant to, add high salt precipitated liquid 250 μ l, Virahol 250 μ l, put upside down repeatedly about 30 times, are put in 10min on ice again.

6. 4 ℃, 12000rpm, centrifugal 10min, supernatant discarded.

7. add 1.2ml 75% ethanol mixing, 4 ℃, the centrifugal 5min of 9045rpm.

8. supernatant discarded, air drying 10min adds 50-80 μ l DEPC dissolution precipitation, and solution is RNA solution, and is subsequent use in-80 ℃ of preservations.

2, cDNA's is synthetic

Adopt 3 ' the Full-RACE test kit (3 '-Full RACE Core Set Ver 2.0) of Dalian TaKaRa company to carry out the synthetic of cDNA, concrete grammar is following:

Press following mixed sample:

RNase Free ddH2O 4.5μl

3’RACE Adaptor(5μM) 1μl

RNA(1.5μg total RNA) 1μl

Behind the mixing, 70 ℃ of sex change 10min, chilling 2min on ice.

Above-mentioned reaction soln 6.5 μ l

dNTP Mixture(10mM each) 1μl

RNase Inhibitor 0.25μl

Reverse Transcriptase M-MLV(RNase H-) 0.25μl

5×M-MLV Buffer 2μl

Behind the mixing, 42 ℃ of reaction 1h, 70 ℃ of reaction 15min, the reverse transcription product after reaction finishes is in-20 ℃ of preservations.

3,3 '-RACE of conotoxin cDNA amplification

With step 2 synthetic cDNA is template, and according to conotoxin peptide A superfamily α conotoxin peptide signal peptide zone design α-CTX outside primers F 1 and inboard primers F 2, with 3 '-RACE outside primer R1, the inboard primer R2 of 3 '-RACE carries out nest-type PRC respectively.

F1：5’-ATGGGCATGCGGATGATGTTC-3’；

F2：5’-CTGTTGGTTGTCTTGGCAACCAC-3’；

R1：5’-TACCGTCGTTCCACTAGTGATTT-3’；

R2：5’-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3’。

Concrete steps:

First round PCR reaction system comprises the cDNA template 3 μ l of reverse transcription, 1 * cDNA Dilution Buffer II7 μ l, upstream primer F1 2 μ l, 3 '-RACE outside primer R1 2 μ l, 10 * LA PCR Buffer II (Mg ²⁺Plus) 5 μ l, TaKaRa LA Taq (5U/ μ l) 0.25 μ l is with the deionized water polishing TV 50 μ l of sterilization.The cycling condition that carries out PCR is 94 ℃ of preparatory sex change 4 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended 50 seconds (barrel-shaped cone shell and password cone shell are 1min), and 30 circulations are extended 10min, 4 ℃ of insulations at 72 ℃ at last.

Second takes turns the PCR reaction system comprises first round PCR product 1 μ l, dNTP Mixture (each 2.5mM) 8 μ l, 10 * LA PCR Buffer II (Mg ²⁺Plus) 5 μ l, the inboard primer R2 of 3 '-RACE 2 μ l, downstream primer F2 2 μ l, TaKaRa LA Taq (5U/ μ l) 0.5 μ l is with the deionized water polishing TV 50 μ l of sterilization.The cycling condition that carries out PCR is 94 ℃ of preparatory sex change 4 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended 50 seconds (barrel-shaped cone shell and password cone shell are 1min), and 30 circulations are extended 10min, 4 ℃ of insulations at 72 ℃ at last.

The PCR product is got 8 μ L point samples, and amplified production separates through 1.0% agarose gel electrophoresis, and the ultraviolet transmission observation analysis is taken a picture under image analyzer.

4, the clone of PCR product and order-checking

Reclaim above-mentioned specific PCR product (300bp-800bp) with glue recovery test kit and be connected with carrier, 6 kinds of PCR products are connected with pGM-T carrier (TIANGEN biotech firm); Transformed into escherichia coli DH5 α utilizes amicillin resistance to select mono-clonal, and 37 ℃, 220rpm shake bacterium; Selecting positive colony through the PCR method again checks order; The PCR reaction system is reaction bacterium liquid 1 μ l, the inboard primer R2 of 3 '-RACE 1 μ l, downstream primer F2 1 μ l; 2 * Taq PCRMaster Mix (TIANGEN biotech firm), 12.5 μ l, ddH ₂O 9.5 μ l, the total reaction system is 25 μ l, and reaction conditions same second is taken turns the cycling condition of PCR, and positive colony checks order, and through the analysis and the comparison of sequence, has obtained the novel α conotoxin peptide sequence of general formula I according to the invention.The novel α conotoxin peptide sequence of general formula I is to infer according to propetide and conotoxin characteristics, and propetide is to infer according to precursor-gene.6 kinds of α conotoxin peptide propeptide sequences are seen table 1, and 6 kinds of α conotoxin peptide propetide polynucleotide encoding sequences are seen table 2.

Table 1 propeptide sequence

Title	Base number	Direction N '-C '
			T-1P	63	MGMRMMFTVFLLVVLATTVVSFTSDRAPDGRNAAAKAFGLITPTVRKGCCSNPACMLKNPNLC*
T-2P	62	MGMRMMFTVFLLVVLATTVISFTSDRASDDGNSAVDLKARTVIMHNCCTRSFCKRIYPDLCS*
			T-3P	64	MGMRMMFTVFLLVVLATTVVSDRASNRENRRASNWNTRIAYIGCCDIPDCYNKNREQCLDESSG*
T-4P	62	MGMRMMFTVFLLVVLATTVVSFTLDRASDGASAAADLVARGIRGNCCMFHTCPIDYSRFNCP*
			T-5P	62	MGMRMMFTVFLLVVLATTVVSFTLDRASDGASAAADLVARGIRGNCCMFHTCPIDYSRFNCP*
T-6P	62	MGMRMMFTVFLLVVLATTVVSFTLDRASDGANAAADLVARGIRGNCCMFHTCPIDYSRFYCP*
			T-7P	60	MGMRMMFTVFLLVVLATTVVSLTLDRASGGRRSGADNMIALLIIRKCCSNPACNRYNKLC*

Polynucleotide (DNA) sequence of 7 kinds of α conotoxin peptide propetides of table 2 coding

Title	Base number	Direction 5 '-3 '
			T-1N	263bp	See the sequence 1 of sequence table
T-2N	400bp	See the sequence 2 of sequence table
			T-3N	246bp	See the sequence 3 of sequence table
T-4N	454bp	See the sequence 4 of sequence table
			T-5N	454bp	See the sequence 5 of sequence table
T-6N	400bp	See the sequence 6 of sequence table
			T-7N	203bp	See the sequence 7 of sequence table

Synthesizing of embodiment 2, alpha-type conus polypeptide

Seven peptide species shown in the synthetic respectively table 3.

The aminoacid sequence of table 3 alpha-type conus polypeptide

Title	Sequence
		T-1	NH 2-GCCSNPACMLKNPNLC*
T-2	NH 2-NCCTRSFCKRIYPDLC*
		T-3	NH 2-GCCDIPDCYNKNREQC*
T-4	NH 2-NCCMFHTCPIDYSRFNC*
		T-5	NH 2-GNCCMFHTCPIDYSRFNC*
T-6	NH 2-GNCCMFHTCPIDYSRFYC*
		T-7	NH 2-RKCCSNPACNRYNKLC

The D-aspartic acid; E-L-glutamic acid; The I-Isoleucine; K-Methionin; The L-leucine; The M-methionine(Met); The N-l-asparagine; The Q-Stimulina; The R-l-arginine; The S-Serine; The T-Threonine; The G-glycocoll; The F-phenylalanine(Phe); The P-proline(Pro); The H-Histidine; The A-L-Ala; Y-tyrosine.T-1 and T-7, the disulfide linkage mode of connection is C1-C3 in each polypeptide, C2-C4.* represent amidation (CONH ₂).

One, polypeptide

(1) synthetic (the adopting Fmoc method synthetic) of T-1

1, peptide resin is synthetic

Use solid phase synthesis technique.On 433A Peptide synthesizer (ABI U.S. application system biotech firm instrument), carry out.The amino acid that uses is Fmoc amino acid (U.S. Advanced Chemtech Company products); The resin that uses comprises DCC (Acros Company products), HOBt (U.S. Advanced Chemtech Company products), NMP (PE Company products), piperidines (Shanghai gill biochemical products), methyl alcohol (homemade analytical pure) and methylene dichloride (homemade analytical pure) as Rink resin (U.S. Advanced Chemtech Company products) and Wang resin (U.S. Advanced Chemtech Company products), the reagent of use.

The amino acid whose Side chain protective group of Fmoc is respectively as follows: the Side chain protective group of His, Cys, Asn, Gln be trityl (Trt); The Side chain protective group of Cys is that trityl (Trt) or Acm, is beneficial to step-by-step oxidation; The Side chain protective group of Arg be 9-phenyl fluorene methyl (Pbf); The Side chain protective group of Trp be tertbutyloxycarbonyl (Boc); The Side chain protective group of Ser, Tyr be the tertiary butyl (tBu); The Side chain protective group of Asp, Glu, Try be tert.-butoxy (OtBu).

Carboxylated or amidation is by the resin decision of adopting, and what T7 used is king's resin, and what T1 to T6 used is the Rink resin, thus the T7 terminal carboxylization, T1 to T6 terminal amideization.

In the reaction system, resin and amino acid whose mol ratio are 1: 5, synthetic at every turn 0.1mmol peptide resin (having Side chain protective group).

2, the cracking of peptide resin

Carry out cracking in the peptide resin that has Side chain protective group (0.1mmol) the adding 10ml lysate with step 1 acquisition; Behind the cracking 3h, boil off most of trifluoroacetic acid under the normal temperature, add cold diethyl ether then and precipitate, filter, the gained deposition is with the dissolving of 5% (quality percentage composition) acetate, and freeze-drying then obtains peptide T-1 (thick peptide).

The composition of lysate: 88 parts by volume trifluoroacetic acids (TFA), 5 parts by volume H ₂O, 2 parts by volume tri isopropyl silanes and 5 parts by volume DTTs (DTT).

(2) other polypeptide is synthetic

Method is synthetic with T-1's, obtains peptide T-2, T-3, T-4, T-5, T-6, T-7 respectively.

Two, the folding and enrichment of polypeptide

Chromatographiccondition: C-18, Calesil ODS-100 (Promptar Company); 5 μ m, Φ 4.6 * 250mm; Column temperature: 25 ℃; Applied sample amount: 100 μ l; Flow velocity: 1ml/min; Detect wavelength: 214nm; Gradient (, being linear gradient elution): 0-1min, acetonitrile 0%-5% in each time period; 1-25min, acetonitrile 5%-70%; 25-28min, acetonitrile 70%-80%.

1, polypeptide is folding

(1) one step jackknife method (air oxidation process)

T-2, T-3, T-4, T-5, T-6 adopt a step jackknife method, and step is following: get the polypeptide that the 40mg step 1 obtains, be dissolved in the NH of 300ml 0.1M ₄HCO ₃In the buffered soln, using ammoniacal liquor to regulate the pH value is 8.2, and the room temperature lower magnetic force stirs 24-48h.Behind folding the end, regulate pH value to 5 with Glacial acetic acid min. 99.5, termination reaction is carried out stratographic analysis.

(2) two step jackknife methods

T-1, T-7 adopt two step jackknife methods, earlier with a pair of sulfydryl in the Acm protection linear peptides, with the other a pair of disulfide linkage of air oxidation process oxidation, and then remove the Acm base, a pair of disulfide linkage that formation is left with the iodine oxidation style.Two step jackknife method concrete steps are following: (1) the first step oxidation is folding: folding oxidation 28h in the 0.1M Tris-HCl buffered soln (pH7.7); HPLC monitoring reaction process; Reaction finishes the back and adds the little acetic acid termination reaction; Enrichment desalination freeze-drying, folding behind the purifying with carrying out next step behind the deionized water dissolving; (2) second step oxidations are folding: the 10mM iodine solution mixes with the product equal-volume of 0.4mg/mL step (1), and lucifuge reaction 10min adds an amount of ascorbic acid solution termination reaction then, and HPLC analyzes, enrichment, freeze-drying.

Stratographic analysis figure behind T-2, T-3, T-4, T-5, the folding 24h of T-6 is as shown in Figure 1; A is the stratographic analysis figure behind the folding 24h of T-2, and B is the stratographic analysis figure behind the folding 24h of T-3, and C is the stratographic analysis figure behind the folding 24h of T-4, and D is the stratographic analysis figure behind the folding 24h of T-5, and E is the stratographic analysis figure behind the folding 24h of T-6.Stratographic analysis figure after T-1, the T-7 the first step and the second step oxidation are folding is as shown in Figure 2; A is the stratographic analysis figure of the T-1 the first step after folding, and B is the stratographic analysis figure of second step of T-1 after folding, and C is the stratographic analysis figure of the T-7 the first step after folding, and D is the stratographic analysis figure of second step of T-7 after folding.The folding RT of the first step of peptide T-1 is 17.828min, and second step, folding RT was 17.699; The RT of peptide T-2 is 17.288min; The RT of peptide T-3 is 10.654min; The RT of peptide T-4 is 15.390min; The RT of peptide T-5 is 15.198min; The RT of peptide T-6 is 15.656min; The folding RT of the first step of peptide T-7 is 11.266min, and second step, folding RT was 11.653.

2, polypeptide enrichment

Get T1 polypeptide solution that 300ml step 1 obtains with Waters Delta Prep 4000HPLC enrichment.Preparative column (C18, Nucleosil, 25mm * 100mm, Dalian materialization institute).Last appearance speed is 2.5ml/min; Water behind the end of the sample (containing 0.1%TFA) desalination, the volume of water are two column volumes (about 140ml), flow velocity 4ml/min; Use 90% acetonitrile (containing 0.1%TFA) and 10% water (containing 0.1%TFA) wash-out then, flow velocity 5ml/min collects elutriant, detects under the 214nm.The elutriant rotary evaporation of collecting is removed most of acetonitrile, obtain 70mg peptide T 1 (thick peptide) after the freeze-drying.

Adopt identical method to obtain the thick peptide of T-2, T-3, T-4, T-5, T-6, T-7 respectively.

Three, the purifying of polypeptide (HPLC method)

In the purifying of polypeptide: the RT of peptide T-1～T-7 is between the 10-14min.

In the analysis of polypeptide: the RT of peptide T-1 is 17.675min; The RT of peptide T-2 is 11.631min; The RT of peptide T-3 is 10.669min; The RT of peptide T-4 is 15.133min; The RT of peptide T-5 is 14.889min; The RT of peptide T-6 is 15.504min; The RT of peptide T-7 is 11.554min.

1, the purifying of polypeptide

Get the thick peptide of T1 of 30mg step 2 preparation, use reversed-phase HPLC to carry out purifying.With water and acetonitrile (TFA that contains 0.1% volumn concentration respectively) is moving phase, adopts the method for gradient elution, prepares with the C-18 semipreparative column.Liquid phase HPLC condition is following:

Semipreparative column: C-18, Kromasil, 10m, 10.0 * 250mm;

Flow velocity: 3ml/min;

Detect wavelength: 214nm;

Column temperature: 25 ℃;

Gradient (, being linear gradient elution) in each time period:

0-1min, acetonitrile 5%-10%;

1-25min, acetonitrile 10%-40%;

25-28min, acetonitrile 40%-100%;

Applied sample amount: 2mg.

Collect target peak, boil off most of acetonitrile, obtain 8mg peptide T 1 (pure peptide) after the freeze-drying.

2, the analysis of polypeptide

Adopt the HPLC method to analyze to the pure peptide that obtains T1, the HPLC condition of analytical pure peptide is following:

Analytical column: C-18, Calesil ODS-100,5 μ m, 4.6 * 250mm;

Column temperature: 25 ℃;

Applied sample amount: 100 μ l;

Flow velocity: 1ml/min;

Detect wavelength: 214nm;

Gradient (, being linear gradient elution) in each time period:

0-1min, acetonitrile 0%-5%;

1-25min, acetonitrile 5%-70%;

25-28min, acetonitrile 70%-80%;

Applied sample amount: 20ug.

Adopt identical method to obtain the pure peptide of T-2, T-3, T-4, T-5, T-6, T-7 respectively.

The stratographic analysis figure of T-1, T-2, T-3, T-4, T-5, T-6, T-7 is as shown in Figure 3.Wherein, A is the stratographic analysis figure of T-1, and B is the stratographic analysis figure that T-2 is, C is the stratographic analysis figure of T-3, and D is the stratographic analysis figure of T-4, and E is the stratographic analysis figure of T-5, and F is the stratographic analysis figure of T-6, and G is the stratographic analysis figure of T-7.The result shows that adopting the T-1, T-2, T-3, T-4, T-5, T-6, the T-7 purity that obtain behind the HPLC method purifying is 70-90%.

The analgesic activities of embodiment 3, alpha-type conus polypeptide is measured

One, the preparation of α-conotoxin peptide Vc1.1

α-conotoxin peptide Vc1.1:GCCSDPRCNYDHPEICONH ₂

1, peptide resin is synthetic

Use solid phase synthesis technique.On 433A Peptide synthesizer (ABI U.S. application system biotech firm instrument), carry out.The synthetic amino acid that uses is protected amino acid (U.S. Advanced Chemtech Company products) as Fmoc; The resin that uses is Rink resin (U.S. Advanced Chemtech Company products), and the reagent of use comprises DCC (Acros company), HOBt (U.S. Advanced Chemtech Company products), NMP (PE company), piperidines (the Shanghai gill is biochemical), methyl alcohol (homemade analytical pure) and methylene dichloride (homemade analytical pure).

The amino acid whose Side chain protective group of Fmoc is respectively as follows: the Side chain protective group of His, Cys be trityl (Trt); The Side chain protective group of Arg be 9-phenyl fluorene methyl (Pbf), the Side chain protective group of Trp be tertbutyloxycarbonyl (Boc); The Side chain protective group of Ser, Tyr be the tertiary butyl (tBu), the Side chain protective group of Asp, Glu be tert.-butoxy (OtBu).

In the reaction system, resin and amino acid whose mol ratio are 1: 5, synthetic at every turn 0.1mmol peptide resin (having Side chain protective group).

2, the cracking of peptide resin

Carry out cracking in the peptide resin that has Side chain protective group (0.1mmol) the adding 10ml lysate with step 1 acquisition; Behind the cracking 3h, boil off most of trifluoroacetic acid under the normal temperature, add cold diethyl ether then and precipitate, filter, the gained deposition is with the dissolving of 5% (quality percentage composition) acetate, and freeze-drying then obtains polypeptide Vc1.1 (thick peptide).

The composition of lysate: 88 parts by volume trifluoroacetic acids (TFA), 5 parts by volume H ₂O, 2 parts by volume tri isopropyl silanes and 5 parts by volume DTTs (DTT).

3, the folding and enrichment of polypeptide

Chromatographiccondition: Kromasil C-18 (Beijing Analytical Instrument Factory) analytical column, 5 μ m, Φ 4.6 * 250mm; Column temperature: 25 ℃; Applied sample amount: 100 μ l; Flow velocity: 1ml/min; Detect wavelength: 214nm; Gradient: 1-30min, acetonitrile 5%-60% (linear gradient elution).

(1) polypeptide is folding

Adopt air oxidation process, get the polypeptide Vc1.1 that 90mg step 2 obtains, be dissolved in the NH of 300ml 0.1M ₄HCO ₃In the damping fluid, using ammoniacal liquor to regulate the pH value is 8.5, and the room temperature lower magnetic force stirs 24-48h.Behind folding the end, regulate pH value to 3.5 with Glacial acetic acid min. 99.5, termination reaction is carried out stratographic analysis.

Stratographic analysis figure behind the folding 24h of Vc1.1 is shown in Fig. 1 F.Main peak is target polypeptides Vc1.1 (RT is 14.365min) among the figure.

(2) enrichment of polypeptide

Polypeptide solution after using the enrichment of preparation liquid phase folding, the concrete operations step is following:

Partly prepare in the liquid phase systems at waters 300E, (Φ 25 * 10mm (pre-column)+Φ 25 * 100mm) uses H to preparative column for C-18,15 μ m ₂Two column volumes of O balance (5ml/min, 20min) after, slowly sample introduction (2.5ml/min) is used H ₂Two column volume (2.5ml/min of O wash-out; More than the 40min) to wash away salt and other small-molecule substances, carry out constant gradient wash-out (5ml/min) with 60% acetonitrile again, the absorbancy at monitoring 214nm place; Receive the polypeptide target peak, preserve the C18 post with 100% acetonitrile at last.The polypeptide solution of collecting obtains Vc1.1 (thick peptide) after the freeze-drying.

4, HPLC method purifying

(1) purifying of polypeptide

Getting the first peptide of Vc1.1 of 30mg step 3 preparation, use reversed-phase HPLC to carry out purifying, is moving phase with water and acetonitrile (TFA that contains 0.1% volumn concentration respectively), adopts the method for gradient elution, prepares with the C-18 semipreparative column.Liquid phase HPLC condition is following:

Semipreparative column: Zorabx 300SB, C-18,10m, Φ 9.4 * 250m;

Flow velocity: 3ml/min;

Detect wavelength: 214nm;

Column temperature: 25 ℃;

Gradient (, being linear gradient elution) in each time period:

0-1min, 0-5% volumn concentration acetonitrile;

1-30min, 5%-60% volumn concentration acetonitrile;

30-32min, 60%-100% volumn concentration acetonitrile;

Applied sample amount: 2mg.

Collect target peak (RT is 13-15min), boil off most of acetonitrile, obtain the pure peptide of 6mg Vc1.1 after the freeze-drying.

(2) analysis of polypeptide

Pure peptide to Vc1.1 adopts the HPLC method to analyze, and the HPLC condition of analytical pure peptide is following:

Analytical column: Kromasil C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6 * 250mm;

Flow velocity: 1ml/min;

Detect wavelength: 214nm;

Column temperature: 25 ℃;

Gradient (, being linear gradient elution) in each time period:

0-1min, 0-5% volumn concentration acetonitrile;

1-30min, 5%-60% volumn concentration acetonitrile;

30-32min, 60%-100% volumn concentration acetonitrile;

Applied sample amount: 50ug.

The stratographic analysis figure of Vc1.1 is (RT is 14.465min) shown in Fig. 3 H.The result shows that the purity that adopts the Vc1.1 that obtains behind the HPLC method purifying is greater than 90%.

Two, make up rat sciatic nerve hemisection model

Utilize rat sciatic nerve hemisection model as the analgesic model.This model be to rat sciatic nerve directly puncture damage, it is quick to bring out intensive machinery pain, autophagy seldom appears in animal, analog neuron source property pain preferably, the surgical procedure simple and fast is a kind of good analgesic model simultaneously.

The SD rat, ♂, body weight 200-220g is provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A.Reference literature (Seltzer et al.Pain; A novel behavioral model of neuropathic paindisorders produced in rats by partial sciatic nerve injury.1990; 43, method 205-218.) makes up sciatic nerve hemisection rat model.Concrete steps are following: rat exposes right sciatic nerves in a thigh section high position under anesthesia and aseptic technique; Under 25 times of reading lenses; Carefully neural dorsal part and surrounding tissue are separated to the far-end of PB and semitendinosus bifurcation in sciatic nerve-trunk, clamped neural dorsal part, a 6-0 silicon throwing linear slit is gone in the nerve trunk with little mosquito forceps; Neural dorsal part 3/1-2/1 is received in the line cover, tightens the line cover then.Muscle and skin are all sewed up with cotton thread.

Perform the operation and use Ugo 37215 type tenderness appearance test rat threshold of pain after seven days, threshold of pain decline 50% above person is regarded as modeling success rat.

Two, utilize the analgesic activities of rat sciatic nerve hemisection model determination alpha-type conus polypeptide to rat

1, the analgesic activities of T-1, T-4, T-5, T-6

The rat that modeling is successful is divided into 6 groups; Every group 8; First group to the 4th group alpha-type conus toxin T-1, T-4, T-5, the T-6 that difference intramuscular injection 3nmol (0.2mL) the foregoing description 2 obtains; The 5th group (positive control) injection 3nmolVc1.1 (0.2mL), the 6th group (negative control) injection 0.2mL saline water.Behind the administration 2.5-3h, the variation of threshold of pain before and after the administration of test rat.Three repetitions are established in experiment, and threshold of pain is got the MV of three repeated experiments of 8 rats.Each experimental group rat threshold of pain raising rate is seen table 4.

The analgesic effect of table 4 alpha-type conus polypeptide

2, the analgesic activities of T-2, T-3, T-7

The rat that modeling is successful is divided into 5 groups; Every group 6; First group to the 3rd group alpha-type conus toxin T-2, T-3, the T-7 that difference intramuscular injection 3nmol (0.2mL) the foregoing description 2 obtains; The 4th group (positive control) injection 3nmol Vc1.1 (0.2mL), the 5th group (negative control) injection 0.2mL saline water.Behind the administration 2.5-3h, the variation of threshold of pain before and after the administration of test rat.Three repetitions are established in experiment, and threshold of pain is got the MV of three repeated experiments of 6 rats.Each experimental group rat threshold of pain raising rate is seen table 5.

The analgesic effect of table 5 alpha-type conus polypeptide

Saline water (NS)	65.8	64.7	-1.42±2.2％
				The Vc1.1 group	66.7	89.8	35.1±9.0％
Peptide T-2	65.0	91.3	40.9±11.1％
				Peptide T-3	52.5	76.6	46.8±10.3％
Peptide T-7	61.7	78.3	26.6±7.2％

The analgesic activities result of different alpha-type conus toxin is as shown in Figure 4.Among Fig. 4, ordinate zou is the raising rate of threshold of pain behind the rat muscle administration 2.5-3h, and X-coordinate is represented different alpha-type conus polypeptides.The analgesia result shows that under identical dosage, compare with the saline water group, T-1, T-2, T-3, T-4, T-5, T-6, T-7 have all improved the threshold of pain of rat significantly.The analgesic activities of T-1, T-2, T-3, T-4 is significantly higher than control peptide Vc1.1.When dosage is 3nmol; T-1 can make the threshold of pain of rat improve 80.4 ± 16.0%; Analgesic effect than Vc1.1 (34.3 ± 6.1%) has promoted about 134%; T-2 can make the rat threshold of pain improve 40.9 ± 11.1%, and T-3 can make the rat threshold of pain improve 46.8 ± 10.3%, and T-4 can make the rat threshold of pain improve 58.9 ± 8.5%.T-5 can make the rat threshold of pain improve 26.9 ± 5.9%, and T-6 can make the rat threshold of pain improve 20.6 ± 4.7%, and T-7 can make the rat threshold of pain improve 26.6 ± 7.2%.

Sequence table

< 110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A

< 120>alpha-type conus polypeptide and application thereof

<130>CGGNARY102150

<160>13

<210>1

<211>263

<212>DNA

< 213>Conus cone shell (Conus)

<400>1

atgggcatgc ggatgatgtt caccgtgttt ctgttggttg tcttggcaac caccgtcgtt 60

tctttcactt cagatcgtgc acctgatggc aggaatgccg cagctaaagc gtttggcctg 120

atcactccga ccgtcaggaa agggtgttgt tccaatcctg cctgtatgtt gaaaaatcca 180

aacctatgtt gaaatcacta gtggaacgac ggtaaatcga attcccgcgg ccgccatggc 240

ggccggagca tgcgactcgc ccc 263

<210>2

<211>400

<212>DNA

< 213>Conus cone shell (Conus)

<400>2

atgggcatgc ggatgatgtt caccgtgttt ctgttgctgt tggttgtctt ggcaaccact 60

gtcatttcct tcacttcaga tcgtgcatct gatgacggga attccgcagt cgacctgaag 120

gctcgtaccg tcataatgca caactgctgt acccgctctt tctgtaagag gatttatcca 180

gatttgtgtt cctgaataag ctgctgctgc aggatcctct gaactacgac atgccgccct 240

ctgcctgacc tgcttcactt tccgtctctt tgtgccacta gaactgaaca actcgatcca 300

ctagactccc acgtcacctc cgtattctga aactacctgg ctttgattgt ctttaatttc 360

tagtcacact tgctattatt acatcgtcca aaaaaaaaaa 400

<210>3

<211>246

<212>DNA

< 213>Conus cone shell (Conus)

<400>3

atgggcatgc ggatgatgtt caccgtgttt ctgttgctgt tggttgtctt ggcaaccact 60

gtcgtttcag atcgtgcatc caatcgggag aaccgcagag catctaactg gaatactcgg 120

attgcctaca taggttgctg tgatattcct gattgttata ataaaaaccg ggagcaatgt 180

cttgacgaaa gttctggatg aaatgcaggg atacttggag aatccagagc atcctaaaaa 240

aaaaaa 246

<210>4

<211>454

<212>DNA

< 213>Conus cone shell (Conus)

<400>4

atgggcatgc ggatgatgtt caccgtgttt ctgttgctgt tggttgtctt ggcaaccacc 60

gtcgtttcct ttactttaga tcgtgcatct gatggcgcca gtgcagcagc cgacctggtc 120

gctcgaggca ttaggggaaa ctgctgtatg tttcatacct gtcccattga ctattcacgt 180

tttaattgtc cttgaataag ctgcaactcc aggccctctg aaccacgaca tgctgccctc 240

tgcctgacct gcttcacttt ccgtcttctg tgccactaga attgatcaac tcgatccact 300

agactcctac tttacctccg tattctgaaa ctacttggct ttgattgtct ttattttcta 360

gtcacacttg ctgttattac atcatccaaa atttaaacaa gaacatgagg gccgtctgtt 420

caaagaaatc gggcaatgac aagaaaaaaa aaaa 454

<210>5

<211>454

<212>DNA

< 213>Conus cone shell (Conus)

<400>5

atgggcatgc ggatgatgtt caccgtgttt ctgttgctgt tggttgtctt ggcaaccacc 60

gtcgtttcct ttactttaga tcgtgcatct gatggcgcca gtgcagcagc cgacctggtc 120

gctcgaggca ttaggggaaa ctgctgtatg tttcatacct gtcccattga ctattcacgt 180

tttaattgtc cttgaataag ctgcaactcc aggccctctg aaccacgaca tgctgccctc 240

tgcctgacct gcttcacttt ccgtcttctg tgccactaga attgatcaac tcgatccact 300

agactcctac tttacctccg tattctgaaa ctacttggct ttgattgtct ttattttcta 360

gtcacacttg ctgttattac atcatccaaa atttaaacaa gaacatgagg gccgtctgtt 420

caaagaaatc gggcaatgac aagaaaaaaa aaaa 454

<210>6

<211>400

<212>DNA

< 213>Conus cone shell (Conus)

<400>6

atgggcatgc ggatgatgtt caccgtgttt ctgttgctgt tggttgtctt ggcaaccact 60

gtcgtttcct ttaccttaga tcgtgcatct gatggcgcca atgcagcagc cgacctggtc 120

gctcgaggca ttaggggaaa ctgctgtatg tttcatacct gtcccattga ctattcacgt 180

ttttattgtc cttgagtaag ctgcagctcc aggccctctg aaccacgaca tgctgccctc 240

tgcctgacct gcttcacttt ccgtcttctg tgccactaga attgatcaac tcgatccact 300

agactcctac tttacctccg tattctgaaa ctacttggct ttgattgtct ttatttttct 360

agtcacactt gctgttatta catcatccaa aaaaaaaaaa 400

<210>7

<211>203

<212>DNA

< 213>Conus cone shell (Conus)

<400>7

atgggcatgc ggatgatgtt caccgtgttt ctgttggttg tcttggcaac cacggtcgtt 60

tccctcactt tagatcgtgc atctggtggc aggagatccg gagccgacaa catgattgct 120

cttctgatca tcagaaaatg ctgttccaat cccgcctgta acaggtataa taaactttgc 180

tgaaactcta gaaaaaaaaa aaa 203

<210>8

<211>16

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>8

Gly Cys Cys Ser Asn Pro Ala Cys Met Leu Lys Asn Pro Asn Leu Cys

1 5 10 15

<210>9

<211>16

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>9

Gly Cys Cys Asp Ile Pro Asp Cys Tyr Asn Lys Asn Arg Glu Gln Cys

1 5 10 15

<210>10

<211>17

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>10

Asn Cys Cys Met Phe His Thr Cys Pro Ile Asp Tyr Ser Arg Phe Asn

1 5 10 15

Cys

<210>11

<211>18

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>11

Gly Asn Cys Cys Met Phe His Thr Cys Pro Ile Asp Tyr Ser Arg Phe

1 5 10 15

Asn Cys

<210>12

<211>18

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>12

Gly Asn Cys Cys Met Phe His Thr Cys Pro Ile Asp Tyr Ser Arg Phe

1 5 10 15

Tyr Cys

<210>13

<211>16

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>13

Arg Lys Cys Cys Ser Asn Pro Ala Cys Asn Arg Tyr Asn Lys Leu Cys

1 5 10 15

Claims (7)

1. following polypeptide derivative: NH ₂-GCCSNPACMLKNPNLC-CONH ₂

2. the encoding sox of following polypeptide: GCCSNPACMLKNPNLC.

3. gene as claimed in claim 2 is characterized in that: the encoding sox of said polypeptide is that the sequence 1 of sequence table is held the dna molecular shown in the 142nd to 189 Nucleotide from 5 '.

4. one kind is used for the analgesic medicine, and its activeconstituents is the described polypeptide derivative of claim 1.

5. the described polypeptide derivative of claim 1 is used for the application of analgesic medicine in preparation.

6. an analgesic agent is the described polypeptide derivative of claim 1.

7. the application of the described polypeptide derivative of claim 1 in the preparation analgesic agent.

Images