CN102286079B - Alpha type conotoxin peptides and applications thereof - Google Patents

Abstract

The invention discloses seven alpha type conotoxin peptides and applications thereof. The peptide I has an amino acid sequence shown as the sequence 7 in a sequence table, and the last amino acid residue is amidated; the peptide II has an amino acid sequence shown as the sequence 8 in the sequence table, and the last amino acid residue is amidated; the peptide III has an amino acid sequence shown as the sequence 9 in the sequence table, and the last amino acid residue is amidated; the peptide IV has an amino acid sequence shown as the sequence 10 in the sequence table, and the last amino acid residue is amidated; the peptide V has an amino acid sequence shown as the sequence 11 in the sequence table, and the last amino acid residue is amidated; the peptide VI has an amino acid sequence shown as the sequence 12 in the sequence table, and the last amino acid residue is amidated; and the peptide VII has an amino acid sequence shown as the sequence 13 in the sequence table. Proved by determination of activity, the seven peptides shown as a general formula I have an analgesic effect, wherein alpha type conotoxin peptides T-1, T-2, T-3 and T-4 have remarkable analgesic activity, and the alpha type conotoxin peptides T5-T7 also have remarkable analgesic activity, and the application value on the aspect of analgesia is achieved.

Description

A kind of alpha-type conus polypeptide and application thereof

The application is that application number is 201010121879.7, the applying date is on March 10th, 2010, denomination of invention is divided an application for the patent application of " 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

Conus gasteropod, the whole world approximately have more than 500 to plant, and spread 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, contain 50-200 active polypeptide in the venom of every kind of cone shell, the contained bioactive peptide of different varieties cone shell is different, even cone shell of the same race is different because of the marine site, its toxin composition also can there are differences, estimate approximately to exist in theory the polypeptide (McIntosh of more than 50,000 kind of different activities, J.M., Jones, R.M. (2001) .Cone venom-from accidental stings to deliberate injection.Toxicon, 39, 1447-1451.Nelson, L. (2004) .Venomous snai ls:One slip, and you ' re dead.Nature, 429, 798-799.).Conotoxin peptide energy specific effect is in the various receptor subtypes of the neurotransmitters such as vagusstoff, and the different kinds of ions passages such as calcium, sodium and potassium, not only can directly as medicine, can also be used for as desirable molecular model the lead compound of developing new drug.Wherein, ω type conotoxin peptide MVIIA is come into the market by the medicine of FDA approval as treatment HIV and terminal cancer pain; The SO-3 that finds from the South China Sea Conus striatus also has very strong analgesic activities (Dai qy 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 sheath, is inconvenient to use.

(α-CTX) belong to conotoxin peptide A superfamily (being divided into A, M, O, P, I, J, S, the 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), formed by 16 amino acid, contain two pairs of disulfide linkage (arrangement mode of disulfide linkage is 1-3,2-4) (Sandall et al.Biochemistry 2003,42,6904-6911.).Studies show that Vc1.1 has shown preferably analgesic activities and had in the neuropathic pain animal model accelerates effect (the Satkunanathan et al.Brain Res 2005 that injured nerve recovers, 1059,149-158), entered 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 alpha-type conus polypeptide, they are significant for the medicine of Development of New Generation neuropathic pain, and these polypeptide can adopt the 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 clinically the large class of peripheral nerve pain two due to nervus centralis pain and peripheral nerve injury.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 central nervous system causes, is common in wound, cerebrovascular disease or multiple sclerosis and the tumour etc. of spinal cord.Peripheral nerve pain is that the factors such as wound, ischemic, compressing, infection, inflammation or metabolism are damaged due to peripheral nerves, as phantom limb pain, postherpetic neuralgia, polyneuritis, diabetic peripheral neuralgia etc.The classical symptom of neuropathic pain comprises paresthesia, hyperpathia and allodynia etc.Medicine commonly used mainly contains the coupling of anticonvulsive drug (carrying western phthalein, oxcarbazepine as Lay), opiates medicine (as morphine) and various thymoleptic clinically, but the problems such as the tolerance of bringing due to toxic side effect and long-term prescription, habituation, not good (the Dray et al of result for the treatment of, Br J Anaesth.2008,101 (1), 48-58; Gilron et al, Expert Opin Emerg Drugs.2007,12 (1), 113-26.).Therefore, find that the analgesic of high reactivity of new generation, low tolerance and non-habituation is very necessary.

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 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 general formula I:

X ₁Be selected from any one amino acid or vacancy in G, R;

X ₂Be selected from any one amino acid in G, N, K;

X ₃Be selected from any one amino acid in S, T, D, M;

X ₄Be selected from any one amino acid in N, R, I, F;

X ₅Be selected from any one amino acid in P, S, H;

X ₆Be selected from any one amino acid in A, F, D, T;

X ₇Be selected from any one amino acid in M, K, Y, P, N;

X ₈Be selected from any one amino acid in L, R, N, I;

X ₉Be selected from any one amino acid in K, I, D, Y;

X ₁₀Be selected from any one amino acid in Y, N;

X ₁₁Be selected from any one amino acid in P, R, S, K;

X ₁₂Be selected from any one amino acid in N, D, E, R, L;

X ₁₃Be selected from any one amino acid or vacancy in L, Q, F;

X ₁₄Be selected from any one amino acid or vacancy in N, Y;

β is selected from amide group or carboxyl;

Above-mentioned letter definition is as follows: D-Asp; E-L-glutamic acid; The I-Isoleucine; K-Methionin; L-Leu; The M-methionine(Met); The N-l-asparagine; The Q-glutamine; The R-arginine; The S-Serine; The T-Threonine; The G-glycine; The F-phenylalanine; The P-proline(Pro); The H-Histidine; The A-L-Ala; Y-tyrosine.

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

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

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

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

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

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

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

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

The aminoacid sequence of 7 polypeptide is as follows:

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 described polypeptide II is specific as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 8 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide II.The preparation method of described polypeptide III is specific as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 9 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide III.The preparation method of described polypeptide IV is specific as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 10 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide IV.The preparation method of described polypeptide V is specific as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 11 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide V.The preparation method of described polypeptide VI is specific as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 12 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide VI.The preparation method of described polypeptide I is specific as follows: the amino acid sequence of chemosynthesis is as shown in the sequence 7 of sequence table and the amidated linear polypeptide of last amino acids residue, be placed in pH7.70.1M Tris-HCl damping fluid oxidation 28h, then add the acetic acid termination reaction, the desalination freeze-drying, then with 10mM iodine solution lucifuge reaction 10min, obtain polypeptide I.The preparation method of described polypeptide VII is specific as follows: the linear polypeptide shown in the sequence 13 of chemosynthesis sequence table, be placed in pH7.70.1M Tris-HCl damping fluid oxidation 28h, then add the acetic acid termination reaction, the desalination freeze-drying, then with 10mM iodine solution lucifuge reaction 10min, obtain polypeptide VII.

The encoding gene of described polypeptide also belongs to protection scope of the present invention.

The encoding gene of described 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 for the analgesic medicine, and its activeconstituents is above arbitrary described polypeptide.

Above arbitrary described polypeptide is also belonging to protection scope of the present invention for the preparation of the application in the medicine of analgesic.

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

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

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

(II) with described alpha-type conus polypeptide or (I) derivative of alpha-type conus polypeptide carry out cyclisation, esterified or PEGization modification, the derivative of the alpha-type conus polypeptide that obtains;

(III) with described alpha-type conus polypeptide or (I) derivative of alpha-type conus polypeptide connect with carrier, the derivative of the alpha-type conus polypeptide that obtains;

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

In 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.

Described replacement can also be " conservative property replacement " or " non-conservation replacement ".Replacement can be an amino acid whose replacement, can be also a plurality of amino acid whose replacements, can be continuous replacement, can be also the replacement that disperses." conservative property replacement ": amino acid, the rare amino acid of nature existence or manually modified amino acid substitution with the one or more amino-acid residue conformations in described alpha-type conus polypeptide are the D-type, suppress active to increase bioavailability and raising; The amino acid of D-type refers to the amino acid relative with the amino acid of 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, as 5-oxylysine, methylhistidine, γ-aminobutyric acid, homoserine etc.; Manually modified amino acid refers to through Hypermethylation, 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 polypeptide are replaced by amino acid of different nature, are perhaps replaced by unconventional amino acid.

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

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

Described 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 as Methionin, halfcystine, perhaps other molecule is joined together to form polymer.

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

The acceptable auxiliary material of pharmaceutics be can add in described medicine, injection, oral preparation, rectum body made 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 were in the situation that intramuscular injection dosage both can make threshold of pain rate improve more than 40% at very low (10nmol/kg), 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 has significant difference with the alpha-type conus polypeptide of present report on amino acid forms.

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 after the folding 24h of T-2, T-3, T-4, T-5, T-6 and Vc1.1; The A:T-2 folding picture; The B:T-3 folding picture; The C:T-4 folding picture; The D:T-5 folding picture; The E:T-6 folding picture; The F:Vc1.1 folding picture.

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

Fig. 3 is the stratographic analysis figure after T-1, T-2, T-3, T-4, T-5, T-6, T-7, 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 in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges 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

Gather 6 kinds of cone shell live bodies from the Xisha Islands, Sanya, Hainan and other places as sample material, adopt respectively the Trizol method to extract total RNA of 6 kinds of cone shells.6 kinds of cone shells: marble cone shell (Conus marmoreus), grand cone shell (Conus imperialis), 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 on freezing in liquid nitrogen and use the mill grinding powder, pouring in the micro-homogenizer of prior ice bath.

2. drawing 1.2ml Trizol adds homogenizer and further the grinding to make cone shell organize cracking to discharge RNA.

3. ground slurries are poured in the centrifuge tube of 1.5ml into the standing 5min of room temperature.

4. add 20% chloroform 240 μ l, after 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, repeatedly put upside down the left and right 30 times, then are put in 10min on ice.

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, saves backup in-80 ℃.

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 as follows:

In following ratio biased sample:

After 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

Take the synthetic cDNA of step 2 as template, 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 II, 7 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) 0.2 μ l is with the deionized water polishing cumulative volume 50 μ l of sterilization.The cycling condition that carries out PCR is 94 ℃ of denaturations 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 cumulative volume 50 μ l of sterilization.The cycling condition that carries out PCR is 94 ℃ of denaturations 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 the test kit above-mentioned freeze-draw method of recovery (300bp-800bp) with glue and be connected with carrier, 6 kinds of PCR products are connected with pGM-T carrier (TIANGEN biotech firm); Transform bacillus coli DH 5 alpha, utilize amicillin resistance to select mono-clonal, 37 ℃, 220rpm shake bacterium, selecting positive colony by 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 PCR Master 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 analysis and the comparison of sequence, has obtained the Novel alpha conotoxin peptide sequence of general formula I of the present invention.The Novel alpha 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.The polynucleotide sequence that 6 kinds of α conotoxin peptide propeptide sequences see Table 1,6 kind of α conotoxin peptide propetide coding sees 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	MGMRMMFTVFLLVVLATTVVSLTLDRASGGRRSGADNMIALLI IRKCCSNPACNRYNKLC*

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 difference synthetic 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

D-Asp; E-L-glutamic acid; The I-Isoleucine; K-Methionin; L-Leu; The M-methionine(Met); The N-l-asparagine; The Q-glutamine; The R-arginine; The S-Serine; The T-Threonine; The G-glycine; The F-phenylalanine; The P-proline(Pro); The H-Histidine; The A-L-Ala; Y-tyrosine.T-1 and T-7, in each polypeptide, the disulfide linkage mode of connection is C1-C3, 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.Carry out on 433A Peptide synthesizer (ABI the U.S. application system biotech firm instrument).The amino acid that uses is Fmoc amino acid (U.S. Advanced Chemtech company product), the resin that uses is Rink resin (U.S. Advanced Chemtech company product) and Wang resin (U.S. Advanced Chemtech company product), and the reagent of use comprises DCC (Acros company product), HOBt (U.S. Advanced Chemtech company product), NMP (PE company product), piperidines (Shanghai gill biochemical products), methyl alcohol (domestic analytical pure) and methylene dichloride (domestic analytical pure).

The amino acid whose Side chain protective group of Fmoc is as follows respectively: 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 determines by the resin that adopts, and what T7 used is king's resin, and what T1 to T6 used is the Rink resin, thus the T7 terminal carboxyl(group), T1 to T6 terminal amide.

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

2, the cracking of peptide resin

The peptide resin with Side chain protective group (0.1mmol) that step 1 is obtained adds and carries out cracking in the 10ml lysate; After cracking 3h, boil off most of trifluoroacetic acid under normal temperature, then add cold diethyl ether to precipitate, filter, the gained precipitation is dissolved with 5% (quality percentage composition) acetic acid, and then freeze-drying 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 DTT (DTT).

(2) other polypeptide is synthetic

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

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 (in each time period, being linear gradient elution): 0-1min, acetonitrile 0%-5%; 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 as follows: get the polypeptide that the 40mg step 1 obtains, be dissolved in the NH of 300ml 0.1M ₄HCO ₃In buffered soln, regulating the pH value with ammoniacal liquor is 8.2, and the room temperature lower magnetic force stirs 24-48h.After folding the end, regulate pH value to 5 with Glacial acetic acid, termination reaction is carried out stratographic analysis.

(2) two step jackknife methods

T-1, T-7 adopt two step foldings for method, first with a pair of sulfydryl in Acm protection linear peptides, with the other a pair of disulfide linkage of air oxidation process oxidation, and then remove the Acm base with the iodine oxidation style, form remaining a pair of disulfide linkage.Two step jackknife method concrete steps are as follows: (1) the first step oxidative folding: folding oxidation 28h in 0.1M Tris-HCl buffered soln (pH7.7), HPLC monitoring reaction process, reaction adds a small amount of acetic acid termination reaction after finishing, enrichment desalination freeze-drying, folding with carrying out next step after deionized water dissolving after purifying; (2) second step oxidative folding: the 10mM iodine solution mixes with the product equal-volume of 0.4mg/mL step (1), and then lucifuge reaction 10min adds appropriate ascorbic acid solution termination reaction, and HPLC analyzes, enrichment, freeze-drying.

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

2, polypeptide enrichment

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

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

Three, the purifying of polypeptide (HPLC method)

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

In the analysis of polypeptide: the retention time of peptide T-1 is 17.675min; The retention time of peptide T-2 is 11.631min; The retention time of peptide T-3 is 10.669min; The retention time of peptide T-4 is 15.133min; The retention time of peptide T-5 is 14.889min; The retention time of peptide T-6 is 15.504min; The retention time 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.Take water and acetonitrile (TFA that contains respectively 0.1% volumn concentration) as moving phase, adopt the method for gradient elution, with the preparation of C-18 semipreparative column.

Liquid phase HPLC condition is as follows:

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

Flow velocity: 3ml/min;

Detect wavelength: 214nm;

Column temperature: 25 ℃;

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

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 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 as follows:

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

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

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

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

Applied sample amount: 20ug.

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

The stratographic analysis figure of T-1, T-2, T-3, T-4, T-5, T-6, T-7 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.Result shows, adopting T-1, the T-2, T-3, T-4, T-5, T-6, the T-7 purity that obtain after 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.Carry out on 433A Peptide synthesizer (ABI the U.S. application system biotech firm instrument).The synthetic amino acid that uses is Fmoc protected amino acid (U.S. Advanced Chemtech company product); the resin that uses is Rink resin (U.S. Advanced Chemtech company product), and the reagent of use comprises DCC (Acros company), HOBt (U.S. Advanced Chemtech company product), NMP (PE company), piperidines (the Shanghai gill is biochemical), methyl alcohol (domestic analytical pure) and methylene dichloride (domestic analytical pure).

The amino acid whose Side chain protective group of Fmoc is as follows respectively: 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 reaction system, resin and amino acid whose mol ratio are 1: 5, at every turn synthetic 0.1mmol peptide resin (with Side chain protective group).

2, the cracking of peptide resin

The peptide resin with Side chain protective group (0.1mmol) that step 1 is obtained adds and carries out cracking in the 10ml lysate; After cracking 3h, boil off most of trifluoroacetic acid under normal temperature, then add cold diethyl ether to precipitate, filter, the gained precipitation is dissolved with 5% (quality percentage composition) acetic acid, and then freeze-drying 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 DTT (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 damping fluid, regulating the pH value with ammoniacal liquor is 8.5, and the room temperature lower magnetic force stirs 24-48h.After folding the end, regulate pH value to 3.5 with Glacial acetic acid, termination reaction is carried out stratographic analysis.

Stratographic analysis figure after the folding 24h of Vc1.1 is as shown in Fig. 1 F.In figure, main peak is target polypeptides Vc1.1 (retention time is 14.365min).

(2) enrichment of polypeptide

Polypeptide solution after using the enrichment of preparation liquid phase folding, concrete operation step is as follows:

In waters 300E partly prepared liquid phase systems, (Φ 25 * 10mm (pre-column)+Φ 25 * 100mm) used H to preparative column for C-18,15 μ m ₂After two column volumes of O balance (5ml/min, 20min), slowly sample introduction (2.5ml/min), use H ₂Two column volume (2.5ml/min of O wash-out, more than 40min) washing away salt and other small-molecule substances, then carry out Gradient elution (5ml/min) with 60% acetonitrile, 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 freeze-drying.

4, HPLC method purifying

(1) purifying of polypeptide

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

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

Flow velocity: 3ml/min;

Detect wavelength: 214nm;

Column temperature: 25 ℃;

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

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 (retention time is 13-15min), boil off most of acetonitrile, obtain the pure peptide of 6mg Vc1.1 after 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 as follows:

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

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 (retention time is 14.465min) as shown in Fig. 3 H.Result shows, adopts the purity of the Vc1.1 that obtains after HPLC method purifying greater than 90%.

Two, build 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, can bring out strong machinery pain quick, autophagy seldom appears in animal, analog neuron source property pain preferably, the surgical procedure simple and fast, be 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, Anovel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury.1990,43, method 205-218.) builds sciatic nerve hemisection rat model.Concrete steps are as follows: rat exposes right sciatic nerves in a thigh section high position under anesthesia and aseptic technique, under 25 times of magnifying glasses, carefully neural dorsal part and surrounding tissue are separated to the far-end of PB and semitendinosus bifurcation in sciatic nerve-trunk, clamp neural dorsal part with little mosquito forceps, a 6-0 silicon scrap prodn. line is sewed up in nerve trunk, neural dorsal part 3/1-2/1 is received in the line cover, then tightens the line cover.Muscle and skin are all sewed up with cotton thread.

Perform the operation and use Ugo 37215 type tenderness instrument test Pain Regulation In The Rat values after seven days, threshold of pain decline 50% above person is considered as modeling success rat.

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

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

The rat of modeling success 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) above-described embodiment 2 obtains, the 5th group (positive control) injection 3nmolVc1.1 (0.2mL), the 6th group (negative control) injection 0.2mL physiological saline.After 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 mean value of three repeated experiments of 8 rats.Each experimental group Pain Regulation In The Rat value increase rate sees 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 of modeling success 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) above-described embodiment 2 obtains, the 4th group (positive control) injection 3nmol Vc1.1 (0.2mL), the 5th group (negative control) injection 0.2mL physiological saline.After 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 mean value of three repeated experiments of 6 rats.Each experimental group Pain Regulation In The Rat value increase rate sees Table 5.

The analgesic effect of table 5 alpha-type conus polypeptide

The analgesic activities result of different alpha-type conus toxin as shown in Figure 4.In Fig. 4, ordinate zou is the increase rate of threshold of pain after rat muscle administration 2.5-3h, and X-coordinate represents different alpha-type conus polypeptides.The analgesia result show, under identical dosage, compare with the physiological saline 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%, promoted approximately 134% than the analgesic effect of Vc1.1 (34.3 ± 6.1%), T-2 can make the Pain Regulation In The Rat value improve 40.9 ± 11.1%, the Pain Regulation In The Rat value that can make T-3 improves 46.8 ± 10.3%, T-4 can make the Pain Regulation In The Rat value improve 58.9 ± 8.5%.The Pain Regulation In The Rat value that can make T-5 improves 26.9 ± 5.9%, T-6 can make Pain Regulation In The Rat value raising 20.6 ± 4.7%, T-7 can make the Pain Regulation In The Rat value improve 26.6 ± 7.2%.

Claims (8)

1. following polypeptide: NH ₂-GCCDIPDCYNKNREQC-CONH ₂

2. polypeptide as claimed in claim 1, it is characterized in that: the preparation method of described polypeptide is as follows: the amino acid sequence of chemosynthesis is dissolved in the NH of 0.1M as shown in the sequence 9 of sequence table and the amidated linear polypeptide of last amino acids residue ₄HCO ₃In damping fluid, regulating the pH value with ammoniacal liquor is 8.2, and magnetic agitation 24-48h obtains polypeptide.

3. the encoding gene of the described polypeptide of claim 1.

4. gene as claimed in claim 3, it is characterized in that: the encoding gene of described polypeptide is as follows: the sequence 3 of sequence table is from the DNA molecular shown in the 133rd to 180 Nucleotide of 5 ' end.

5. one kind is used for the analgesic medicine, and its activeconstituents is the described polypeptide of claim 1 or 2.

6. the described polypeptide of claim 1 or 2 is for the preparation of the application in the medicine of analgesic.

7. an analgesic agent, be the described polypeptide of claim 1 or 2.

8. the application of the described polypeptide of claim 1 or 2 in the preparation analgesic agent.

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