CN1237584A - Conotoxin peptide - Google Patents

Abstract

The present invention discloses 14 kinds of conotoxin peptides found from brocade cone shell and line cone shell collected from Hainan Island of China, belonging to the field of biological technology. Said invention includes the following steps: adopting cDNA clone method to obtain cDNA sequence (SeqxN) of conotoxin, using cDNA sequence to infer the precursor peptite sequence (Seqxp) of conotoxin, and using the precursor peptide sequence to infer the mature conotoxin peptide sequence (Seqx), and these mature conotoxin peptides can be obtained by means of chemical synthesis or gene in vitro expression measure. The above-mentioned 14 kinds of new toxins possess their respective unique receptor specificity and affinity, so that they possess the value in research of receptor, can be used as condidate and leading medicinal material for developing new medicine, and the toxin 11 possesses the advantages of strong pain-relieving activity and small tolerance, etc..

Description

Conotoxin peptide

The invention discloses 14 kinds of conotoxin peptides, particularly 14 kinds of conotoxin peptides finding picture-weaving in silk cone shell of gathering from China Hainan Island and the strain line cone shell belong to biological field.

Cone shell belongs to Mollusca, Gastropoda, Probranchia, Conidae, Conus.Present known cone shell kind has 500 kinds approximately, and China has 70 kinds approximately.Conotoxin belongs to neurotoxin mostly, strong toxicity, and effect is rapid, and the toxic component complexity in each cone shell venom is various.From different cone shells, be separated to about 40 kinds of conotoxins at present, can be divided into following six classes according to the acceptor that it acted on:

The Alpha conotoxin acts on the n-acetylcholine receptor

The Omega conotoxin acts on the calcium channel acceptor

The Delta conotoxin acts on the sodium-ion channel acceptor

The M/O conotoxin acts on sodium, calcium channel acceptor

The Miu conotoxin acts on the sodium-ion channel acceptor

The Kappa conotoxin acts on the potassium-channel acceptor

Above-mentioned conotoxin is made up of 10～30 amino acid on sequence usually, has conservative halfcystine to arrange, and contains 2 to 3 pairs of disulfide linkage usually.The precursor peptide of normally being made up of 70～80 amino acid after the conotoxin translation obtains ripe toxin peptide through translation post-treatment such as excision signal peptide sequence and former peptide sequence.Though conotoxin all has similar space structure and disulfide linkage framework, but differ an amino acid on its sequence and just may possess new receptor-specific, meaning in acceptor research provides a kind of new instrument, and a kind of new drug candidate and lead drug are provided on new drug development.Conotoxin except in acceptor research as important instrument; also on the clinical treatment He in the medical diagnosis on disease application prospect is being arranged, these application comprise analgesia, ischemia neuroprotective, increase that neurotransmitter under the pathological state discharges, the treatment of small cell lung cancer and detection, as vegetable insecticide etc.

The objective of the invention is to the 14 kinds of conotoxin peptides and the application thereof of discovery open picture-weaving in silk cone shell (Conustextile) of gathering from China Hainan Island and the strain line cone shell (Conus striatus).

The object of the present invention is achieved like this:

A. sequence:

The method that adopts cDNA to clone is obtained the cDNA sequence (SeqxN) of conotoxin, infer the precursor peptide sequence (Seqxp) that conotoxin by the cDNA sequence, infer by the precursor peptide sequence ripe conotoxin peptide sequence (Seqx) that these ripe toxin peptides can obtain through chemosynthesis or gene vivoexpression means; The toxin sequence of 14 kinds of conotoxins among the present invention, the cDNA sequence of toxin and the precursor peptide sequence of toxin are as follows respectively:

Seq1:PECCSDPRCNSSHPELCG*

* represent that this toxin carboxyl terminal is by amidation Seq1N:ATGGGCATGCGGATGATGTTCATCGTGTTTCTGTTGGTTGTCTTGGC

AACCACTGTCGTTTCCTCCACTTCAGGTCGTCGTGCATTTCATGGCA

GGAATGCCGCAGCCAAAGCGTCTGGCCTGGTCAGTCTGACTGACAGG

AGACCAGAATGCTGTAGTGATCCTCGCTGTAACTCGAGTCATCCAGA

ACTTTGTGGTGGAAGACGCTGASeq1p:GMRMMFIVFLLVVLATTVVSSTSGRRAFHGRNAAAKASGLVSLTDRR

PECCSDPRCNSSHPELCGGRRSeq2?:PECCSHPACNVDHPEICRSeq2N:ATGGGCATGCGGATGATGTTCACCGTGTTTCTCTTGGTTGTCTTGGC

AACCACCGTCGTTTCCTTCACTTCAGGTCGTCGTACATTTCATGGCA

GGAATGCCGCAGCCAAAGCGTCTGGCCTGGTCAGTCTGACTGACAGG

AGACCAGAATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCAGA

AATTTGTCGTTGAAGACGCTGASeq2P:MGMRMMFTVFLLVVLATTVVSFTSGRRTFRGRNAAAKASFLVSLTDR

RPECCSHPACNVDHPEICRSeq3?:CLDAGEVCDIFFPTCCGYCILLFCASeq3N:ATGAAACTGACGTGTGTGATGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGCCACGGCTGATGACTCCAGCAATGGATTGGGGAATC

TTTTTTTGAAGGCACATCACGAAATGAAGAACCCCGAAGCCTCTAAG

TTGAACGAGAGGTGCCTTGATGCTGGTGAAGTTTGTGATATTTTTTT

TCCAACATGCTGCGGCTATTGCATTCTTCTTTTCTGCGCATAASeq3P:MKLTCVVIVAVLFLTAWTFATADDSSNGLGNLFLKAHHEMKNPEASK

LNERCLDAGEVCDIFFPTCCGYCILLFCASeq4?:YDCEPPGNFCGMIKIGPPCCSGWCFFACASeq4N:ATGAAACTGACGTGCGTGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGTGCCTCACTCCAGCAATGCATTGGAGA

ATCTTTATCTGAAGGCACGTCACGAAATGGAAAACCCTGAAGCCTCT

AAATTGAACACGAGATACGACTGCGAACCTCCTGGAAATTTTTGTGG

CATGATAAAAATTGGGCCGCCTTGCTGCAGTGGCTGGTGCTTTTTCG

CCTGCGCCTAASeq4P:MKLTCVVIVAVLFLTAWTFVTAVPHSSNALENLYLKARHEMENPEAS

KLNTRYDCEPPGNFCGMIKIGPPCCSGWCFFACASeq5?:NYCQEKWDYCPVPFLGSRYCCDGLFCTLFFCASeq5N:ATGAAACTGACGTGCGTGGTGATCGTTGCTGTGCTGTTCTTGACAGC

CTGGACGCTAGTCATGGCTGATGACTCCAACAATGGACTGGCGAATC

TTTTTTCGAAATCACGTGATGAAATGGAGGACCCTGAAGCTGCTAAA

TTGGAGAAAAACTATTGCCAAGAAAAATGGGATTATTGTCCAGTACC

GTTCTTGGGATCGAGGTATTGCTGCGATGGCTTATTCTGTACTCTTT

TCTTCTGCGCTTGASeq5P:MKLTCVVIVAVLFLTAWTLVMADDSNNGLANLFSKSRDEMEDPEAAK

LEKNYCQEKWDYCPVPFLGSRYCCDGLFCTLFFCASeq6?:CYDGGTSCDSGIQCCSGWCIFVCFSeq6N:ATGAAACTGACGTGTGTGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGTGCCTCACTCCAGCAATGCGTTGGAGA

ATCTTTATCTGAAGGCACATCATGAAATGAACAACCCCGAAGCCTCT

GAATTGAACAAGAGGTGCTATGATGGTGGGACAAGTTGCGACTCTGG

AATCCAATGCTGCAGTGGCTGGTGCATTTTCGTCTGCTTCTAASeq6P:MKLTCVVIVAVLFLTAWTFVTAVPHSSNALENLYLKAHHEMNNPEAS

ELNKRCYDGGTSCDSGIQCCSGWCIFVCFSeq7?:CYDSGTSCNTGNQCCSGWCIFVCLSeq7N:ATGAAACTGACGTGCATGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGCGCCTCACTCCAGCAATGCGTTGGAGA

ATCTTTATCTGAAGGCACATCATGAAATGAACAACCCCGAAGCCTCT

GAATTGAACAACAGGTGCTATGATAGTGGGACAAGTTGTAACACTGG

AAACCAATGCTGCAGTGGCTGGTGCATTTTCGTCTGCCTCTAASeq7P:MKLTCVVIVAVLFLTAWTFVTAAPHSSNALENLYLKAHHEMNNPEDS

ELNKRCYDSGTSCNTGNQCCSGWCIFVCLSeq8?:CVPYEGPCNWLTQNCCDATCVVFWCLSeq8N:ATGAAACTGACGTGCATGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCCATCACCAGCAATGGATTGGAGAATCTTT

TTCCGAATGCACATCACGAAATGAAGAACCCTGAAGCCTCTAAATTG

AACAAGAGGTGCGTTCCATACGAGGGCCCTTGTAATTGGCTTACACA

AAACTGCTGTGATGCAACATGCGTAGTATTTTGGTGCCTATAASeq8P:MKLTCVVIVAVLFLTAWTFVTAITSNGLENLFPNAHHEMKNPEASKL

NKRCVPYEGPCNWLTQNCCDATCVVFWCLSeq9?:CRPSGSPCGVTSICCGRCYRGKCT*

* represent that this toxin carboxyl terminal is by amidation Seq9N:ATGAAACTGACGTGCGTGGTGATCGTCGCCGTGCTGCTCCTGACGGC

CTGTCAACTCATCACAGCTGAGGACTCTAGAGGTGCGCAGAAGCATC

GTACCCTGCGGTCGACCGCCAGACGCTCCAAGTCCGAGTTGACTACG

AGATGCAGGCCTAGTGGATCCCCCTGTGGTGTTACTAGTATATGCTG

TGGTAGATGCTATAGGGGTAAATGTACGTAGSeq9P:MKLTCVVIVAVLLLTACQLITAEDSRGAQKHRTLRSTARRSKSELTT

RCRPSGSPCGVTSICCGRCYRGKCTSeq10:CRPSGSPCGVTSICCGRCSRGKCT*

* represent that this toxin carboxyl terminal is by amidation Seq10N:ATGAAACTGACGTGCATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGAGGACTCTAGAGGTACGCAGAAGCA

TCGTACCCTGCGGTCGACCGCCAGACGCTCCAAGTCCGAGTTGACT

ACGAGATGCAGACCTAGTGGATCCCCCTGTGGTGTTACTAGTATAT

GCTGTGGTAGGTGCTCTAGGGGTAAATGTACGTAGSeq10P:MKLTCVVIVAVLLLTACQLITAEDSRGTQKHRTLRSTARRSKSELT

TRCRPSGSPCGVTSICCGRCSRGKCTSeq11?:CKAAGKPCSRIAYNCCTGSCRSGKC*

* represent that this toxin carboxyl terminal is by amidation Seq11N:ATGAAACTGACGTGTATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTACCCTGCGGTCGAAGACCAAACTCTCCATGTCGACTCGCTGC

AAGGCTGCAGGAAAACCATGCAGTAGGATTGCGTATAACTGCTGCA

CCGGTTCTTGCAGATCAGGTAAATGTGGCTGASeq11P:MKLTCVVIVAVLLLTACQLITADDSRGTQKHRTLRSKTKLSMSTRC

KAAGKPCSRIAYNCCTGSCRSGKCGSeq12?:ATDCIEAGNYCGPTVMKICCGFCSPYSKICMNYPKNSeq12N:ATGAAACTGACGTGCATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTTCCCTGAGGTCGACCACCAAAGTCTCCAAGGCGACTGACTGC

ATTGAAGCCGGAAATTATTGCGGACCTACTGTTATGAAAATCTGCT

GCGGCTTTTGCAGTCCATACAGCAAAATATGTATGAACTATCCCAA

AAATTGASeq12P:MKLTCMVIVAVLLLTACQLITADDSRGTQKHRSLRSTTKVSKATDC

IEAGNYCGPTVMKICCGFCSPYSKICMNYPKNSeq13?:STSCMEAGSYCGSTTRICCGYCAYFGKKCIDYPSNSeq13N:ATGAAACTGACGTGCGTGATGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTTCCCTGAGGTCGACCACCAAAGTCTCCAAGTCGACTAGCTGC

ATGGAAGCCGGATCTTATTGCGGCTCTACTACGAGAATCTGCTGCG

GTTATTGCGCTTATTTCGGCAAAAAATGTATTGACTATCCCAGCAA

CTGASeq13P:MKLTCVVIVAVLLLTACQLITADDSRGTQKHRSLRSTTKVSKSTSC

MEAGSYCGSTTRICCGYCAYFGKKCIDYPSNSeq14?:DGCSSGGTFCGIHPGLCCSEFCFLWCITFIDSeq14N:ATGAAACTGACGTGTGTGATGATCGTTGCTGTGCTGTTCTTGACCA

CTTGGACATTCGTCACGGCTGATGACTCCAGATATGGATTGAAGAA

TCTTTTTCCGAAGGCACGTCATGAAATGAAGAACCCCGAAGCCTCT

AAATTGAACAAGAGAGATGGGTGCTCTAGTGGTGGTACATTTTGTG

GCATCCATCCAGGACTCTGCTGCAGCGAGTTTTGCTTTCTTTGGTG

CATAACATTTATTGATTGASeq14P:MKLTCVVIVAVLFLTTWTFGTADDSRYGLKNLFPKARHEMKNPEAS

KLNKRDGCSSGGTFCGTHPGLCCSEFCFLWCITFID

In above-mentioned 14 kinds of conotoxins, Seq1～Seq8 is from the picture-weaving in silk cone shell, and wherein Seq1 and Seq2 belong to the Alpha conotoxin; Seq9～Seq14 is from the strain line cone shell, and wherein Seq9～Seq11 belongs to the Omega conotoxin.

B. use:

Toxin Seq11 has analgesic activities, can be used as a kind of analgesic.

The conotoxin peptide that possesses above-mentioned sequence has following advantage:

(1) owing to differing an amino acid, toxin sequence just may produce new receptor-specific, thereby above-mentioned 14 kinds of new toxin all have its unique receptor-specific and avidity, having value in the research of acceptor, is the drug candidate and the lead drug of new drug development;

(2) toxin Seq11 plays a role by the blocking-up calcium channel, and it is strong to have an analgesic activities, the advantage that tolerance is little.

The present invention is described further below in conjunction with embodiment and accompanying drawing.

Fig. 1 is the plasmid map of the expression vector pTrxFus of employing in the embodiment of the invention 3.Wherein:

PTrxFus-plasmid title pL-people phage pL promotor

ATG-initiator codon Thioredoxin-Trx

Ek-enteropeptidase recognition site term-transcription termination signal sequence

The COIE1-escherichia coli plasmid duplicates control sequence

β-Lactamase-β-Nei Xiananmei (ampicillin resistance gene)

The reverse transcription polymerase chain reaction (RT-PCR) of embodiment 1. picture-weaving in silk cone shell Alpha conotoxin cDNA

Picture-weaving in silk Conus food spiral shell cone shell shows strong toxicity to mollusk, and the people is also had lethal effect, and the example of biting of people being poisoned to death because of the picture-weaving in silk cone shell was once arranged in history.People such as Newcomb R once adopted traditional biochemical separation means to find to have tens of kinds of toxin in the picture-weaving in silk cone shell venom, but they fail to illustrate its sequence and biological activity.The toxin of finding from the picture-weaving in silk cone shell at present has Tx I A, Tx I B, Tx II A, Tx VI A, Tx VII, Tx VII A, KK-1, KK-2, convulsions peptide, itch peptide etc., and these belong to Omega, Delta conotoxin respectively, therefrom do not find the Alpha conotoxin as yet.The Alpha conotoxin be owing to can distinguish N﹠M type acetylcholine receptor (nAch-R), thereby plays an important role in the research of nAch-R.In addition the Alpha conotoxin can in conjunction with and block the nAch-R on small cell lung cancer cell surface, in the diagnosis of small cell lung cancer with in treating potential using value is arranged.

Studies show that isolating Alpha conotoxin and Kappa A conotoxin its signal coding sequence of cDNA and 3 ' end non-translational region are very conservative from different cone shells, thereby this two classes conotoxin is called A-family conotoxin, its cDNA can adopt the primer at these conservative region designs to increase.To separate new Alpha conotoxin sequence the picture-weaving in silk cone shell and study its using value in order to produce from China South Sea, we dissect 12 and pick up from also-70 picture-weaving in silk cone shell of ℃ preservation (mean size: long 7～8cm of Sanya, China Hainan, wide 3～4cm) and separate its poison pipe, obtain the separation that the malicious pipe of 6 grams is used for mRNA altogether.

Adopting the Fast Track 2.0Kit mRNA separating kit of Invitrogen company to extract poison pipe mRNA, is that primer carries out reverse transcription (concrete operations are shone the test kit specification sheets and carried out) with T18 with the TimeSaver cDNA synthetic agent box of Pharmacia Biotech company.With carrying out the Alpha conotoxin cDNA that pcr amplification obtains the picture-weaving in silk cone shell with the signal peptide encoding part paired primer P1 (5 ' TCTGCGAATGGGCATGCGGATGATGTT3 ') of A-family conotoxin with the primer P2 (5 ' TGCTCCAACGTCGTGGTTCAGAGGGTC3 ') of 3 ' end untranslated portion paired respectively.

Use Taq archaeal dna polymerase (Huamei Bio-Engrg Co.) and Vent archaeal dna polymerase (BioLabs company) to be mixed into the performing PCR amplification to reduce mutation rate with 100: 1 ratios.PCR is reaction conditions routinely, and reaction volume is 20 μ l, and loop parameter is 94 ℃, behind the sex change 3min, and 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s, totally 25 circulations.The PCR product that obtains is connected to after phosphorylation is handled among processing of Sma I and the dephosphorylized cloning vector pUC18 for sequential analysis with the Wizard PCR Preps test kit purifying of Promega company.Sequential analysis is carried out on the 373A type dna sequencing instrument of Applied Biosystem company.

Respectively from the recombinant plasmid that the two batches of PCR products form at random the picking recon carry out sequential analysis, measured 9 clones from wherein a collection of, another batch measured 10 clones, the result has obtained two kinds of different cDNA sequences, is respectively Seq1N and Seq2N.Coding Seq1N's respectively has 7 among the two crowdes of clones, and coding Seq2N's has 2 and 3 clones respectively.Infer the sequence Seq1p and the Seq2p of the conotoxin precursor peptide that obtains by Seq1N and Seq2N.According to the translation post-treatment rule of known conotoxin, infer that Seq1p and Seq2p obtain ripe Alpha conotoxin Seq1 and Seq2 respectively through the translation post-treatment, the arrangement mode of its halfcystine is CCX4CX7C, belongs to 4/7 hypotype Alpha conotoxin.

Embodiment 2. obtains O-superfamily conotoxin sequence from the strain line cone shell

Have an appointment at the South Sea 70 kinds of cone shells of China distribute, but kind of ichthyophagy cone shell and quantity are few.The strain line cone shell is one of the minority ichthyophagy cone shell kind that can adopt of China Hainan, and its venom is also toxic to the mankind.Adopt biochemical separation means therefrom to find two kinds of Omega conotoxins (S VI A and S VI B), three kinds of Alpha conotoxin (S I at present, S I A, the S II), a kind of Kappa A conotoxin (S IV A) and a kind of cone shell pressurization peptide, these toxin still can not be applied to clinical at least at present.

So far document has been reported the cDNA sequence of 7 kinds of conotoxins, though they belong to Omega respectively, and Delta, Kappa and M/O conotoxin, its signal peptide encoding part is very conservative, thereby has the people that these a few class conotoxins are collectively referred to as the O-superfamily conotoxin.The characteristics that the conservative property of O-superfamily conotoxin cDNA signal peptide encoding part and eukaryotic mrna all have the polyA tail provide convenience for we utilize the O-superfamily conotoxin cDNA of RACE (Rapid Amplification ofcDNAEnds, rapid amplifying cDNA end) method amplification strain line cone shell.We adopt the RACE method to seek new O-superfamily conotoxin sequence from strain line cone shell and picture-weaving in silk cone shell.

Adopt the method among the embodiment 1 to extract poison pipe mRNA from 25 strain line cone shell poison pipes, using the same method with P3 (GGCCACGCGTCGACTAGTACT (18) is N (A/G/C)) is that primer carries out the synthetic cDNA of reverse transcription.Getting 0.5 μ l reverse transcription product is template, add 20ng5 ' end PCR bow thing P4 (TATGAAA CTGACGTG (C/T) is TG (A/G) TGATCGT (A/G)) and 20ng 3 ' end PCR primer P5 (GGCCACGCGTCGACTAGTAC), reaction conditions carries out pcr amplification with Taq archaeal dna polymerase (Huamei Bio-Engrg Co.) routinely, reaction volume is 20 μ l, reacts on 9600 type thermal cyclers of Perkin Elmer company.After loop parameter is 94 ℃ of sex change 3min, 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s, totally 25 circulations.

Pcr amplification product is connected in the pGEMR-T carrier of Promega company behind the Wizard of Promega company PCR Preps test kit purifying, chooses white colony on the ammonia benzyl agar plate of X-gal and carries out PCR and identify containing behind the transformed into escherichia coli DH5 α.Being accredited as the male bacterium colony adopts the QIAprep Spin Miniprep test kit purifying cloned plasmids of QIAGEN company for sequential analysis after increasing.The order-checking of plasmid DNA adopts the 373A dna sequencing instrument of AppliedBiosystem company to carry out the automatic sequence analysis.By about 30 recombinant plasmids that obtain are carried out dna sequence analysis, from the strain line cone shell that gather in China Hainan, be separated to 8 kinds of conotoxin sequences altogether, wherein 6 kinds are the new O-superfamily conotoxin that do not appear in the newspapers as yet (Seq9～Seq14).

Adopting uses the same method obtains 6 kinds of new O-superfamily conotoxins (Seq3～Seq8) from the picture-weaving in silk cone shell.

Embodiment 3. conotoxin Seq3 are at expression in escherichia coli

Vivoexpression is one of method that obtains conotoxin, especially when the chemosynthesis of toxin (for example, Seq3 is too much owing to hydrophobic amino acid content among the present invention, and the little peptide of synthetic is water insoluble) more at need.Because conotoxin has only 10～30 amino acid usually, thereby amalgamation and expression is a kind of selection easily.The fusion protein molecule amount is moderate, detects easily and purifying, and help the folding of toxin peptide under the fusion state.Fusion rotein behind the purifying can discharge needed conotoxin through the cutting of sequence-specific proteolytic enzyme.

In the present embodiment by merging to be implemented in expression in escherichia coli toxin Seq3 with escherichia coli thioredoxin (TrxA).The expression vector that adopts is the pTrxFus of Invitrogen company.This plasmid map such as Fig. 1 have ampicillin resistance gene, plasmid replication control sequence ColE1 and are used to insert the multiple clone site of external source goal gene.Multiple clone site is inserted goal gene with suitable frame and can be obtained fusion rotein in the downstream of Trx TrxA gene.

At first from the cloned plasmids that contains Seq3N, carry out pcr amplification,, handled 2 hours for 37 ℃ with Sal I enzyme to obtain the cDNA fragment of coding Seq3 mature peptide toxin with primer P6 (TGCCTTGATGCTGGTGAA) and primer P5.Expression vector pTrxFus is cut the processing back with Kpn I enzyme scabble, handled 2 hours for 37 ℃ with Sal I enzyme again with the T4 archaeal dna polymerase.The cDNA fragment of the carrier of above-mentioned processing and coding Seq3 mature peptide toxin is connected at 4 ℃ with the T4 dna ligase spends the night.To connect the competent cell of product transformed into escherichia coli GI724, bacterium colony carries out PCR with P5 and P6 and identifies that positive bacterium colony promptly contains our needed expression plasmid pTrxFus/Seq3.The special cleavage site that contains an enteropeptidase in the fusion rotein between TrxA and the Seq3 can obtain toxin peptide Seq3 with enteropeptidase effect fusion rotein from fusion rotein.

The intestinal bacteria GI724 that will contain expression plasmid pTrxFus/Seq3 increases in rich medium and (contains 1 * M9 salt, 2% Casamino Acids, 1% glycerine, 1mmol/L MgCl ₂, 100 μ g/ml penbritins).Cultivate OD600 for 30 ℃ and added tryptophane at 0.5 o'clock to 100 μ g/ml and transfer to 42 ℃ and carry out inducing culture (substratum contains 1 * M9 salt, 0.2%Casamino Acids, 0.5% glucose, 1mmol/L MgCl ₂, 100 μ g/ml penbritins), induce and carry out the SDS-PAGE analysis after 3 hours.Bacterium after the result induces band occurs expressing in the position of about 15KD.Expression product can obtain the Seq3 toxin peptide with the enteropeptidase cutting behind renaturation and preliminary purification.

The chemosynthesis of embodiment 4. conotoxin Seq11

Chemosynthesis is a kind of method easily and efficiently that obtains conotoxin.Adopt the solid state chemistry synthetic method to synthesize toxin Seq11.Initial Rink resin 0.14g (0.1mmol), Fmoc-amino acid 0.5mmol, activator HOBt-BCC, synthetic on 433A (PE company) Peptide synthesizer, get peptide-resin 0.58g.

Above-mentioned peptide-resin is placed lysate, and (contain 0.75g phenol, 0.25ml 1, the 2-3-mercaptoethanol, 0.5ml thioanisole, 0.5ml distilled water, 10ml trifluoroacetic acid) cracking is 2 hours in, filters, the trifluoracetic acid washing, lysate is evaporated to 2ml, adds 100ml anhydrous diethyl ether precipitation, filter, precipitation is dissolved with 20% acetate, gets the 230mg peptide after the freeze-drying.

The freeze-drying peptide is dissolved with 10% acetate, use the G-25 chromatography, 5% acetate wash-out is removed the cracking small molecules, collects the target peptide freeze-drying.

Above-mentioned target peptide is dissolved in the NH4Ac damping fluid of 0.1mol/L (pH8.0 contains the 1mmol/L halfcystine, 1mmol/L EDTA), peptide concentration 0.2mg/ml, 10 ℃ folding 48 hours, the HPLC purifying is used in freeze-drying.Purification column is Zorbax 300 SB-C18,9.4 * 250mm.Gradient is 1～30 minute, 8～28%B solution (3ml/min) (A solution is 0.1%TFA, and B is 100% acetonitrile, contains 0.1%TFA).

The analgesic activities of embodiment 5. toxin Seq11 is measured

Measure the analgesic activities of the toxin peptide Seq11 of chemosynthesis with the mouse hot plate method.Mouse selection body weight is the female mice about 20g, random packet, 5 every group.The contrast of intracerebral injection Seq11, M VII A or physiological saline is measured the threshold of pain of mouse on 55 ℃ of hot plates respectively at different time after the administration, licks the indication that metapedes or metapedes are dithered as pain reaction with mouse.For avoiding mouse to come to harm because of crossing long thermal stimulus, surpass still do not have pain reaction in 60 seconds stop thermal stimulus immediately.When having only indivedual mouse threshold of pain to surpass 60 seconds in one group, when calculating the average threshold of pain in 60 seconds.The result shows (table 1), still has significant analgesic activities when high dosage after (60ng/ mouse) administration in 4 hours.During the 20ng/ mouse, analgesic activities was arranged after the administration in 30 minutes.The analgesic activities of Seq11 is suitable with the analgesic activities of the M VII A that is carrying out clinical experimental study abroad.

Table 1. hot plate method is measured the analgesic activities of toxin seq11

	The threshold of pain (second), basis	30min after the administration (second)	90min after the administration (second)	150min after the administration (second)	240min after the administration (second)
						Seq11 (60ng/ only)	13±1.2	?＞60(5)	????＞60(4),59	????＞60(4),39	????51.8±5.4
M VII A (60ng/ only)	10.2±3.6	?＞60(3),54,42	????＞60(2),34,24, ????37	????＞60(2),36,15, ????32	????37±19.9
Seq11 (30ng/ only)	13.6±6.5	?49.6±12.5	????39.6±14.3	????38.2±19.9	????29.4±17.9
						M VII A (30ng/ only)	10.8±5.2	?46.4±11.8	????26±10.4	????23.2±6.9	????19.6±3.6
	The threshold of pain (second), basis	30min after the administration (second)	60min after the administration (second)	120min after the administration (second)
						Seq11 (20ng/ only)	16±5.2	?43±11.3	????16.2±5.0	????15±5.6
M VII A (20ng/ only)	12.6±5.8	?36±22.6	????13.8±4.8	????12±3.3
Seq11 (10ng/ only)	11.6±5.2	?23.4±3.1	????20±6.2	????15.8±6.6
						M VII A (10ng/ only)	9.8±1.9	?20.0±5.0	????12.6±6.5	????11.4±3.9
						Physiological saline	11.4±2.3	?11.8±2.6	????11.8±2.7	????11.2±1.5

Annotate:＞60 (n), x ... y represents to have in this group n mouse threshold of pain greater than 60 seconds, and all the other several threshold of pain are respectively x ... y second.

Table 1

Claims (1)

1. the invention discloses 14 kinds of conotoxin peptides finding the picture-weaving in silk cone shell of gathering from China Hainan Island and the strain line cone shell, be to adopt cDNA clone's method to obtain the cDNA sequence (SeqxN) of conotoxin, infer the precursor peptide sequence (Seqxp) that conotoxin by the cDNA sequence, infer by the precursor peptide sequence ripe conotoxin peptide sequence (Seqx) that these ripe toxin peptides can obtain through chemosynthesis or gene vivoexpression means; It is characterized in that:

A. sequence:

The toxin sequence of 14 kinds of conotoxins among the present invention, the cDNA sequence of toxin and the precursor peptide sequence of toxin are as follows respectively:

Seq1:PECCSDPRCNSSHPELCG*

* represent that this toxin carboxyl terminal is by amidation Seq1N:ATGGGCATGCGGATGATGTTCATCGTGTTTCTGTTGGTTGTCTTGGC

AACCACTGTCGTTTCCTCCACTTCAGGTCGTCGTGCATTTCATGGCA

GGAATGCCGCAGCCAAAGCGTCTGGCCTGGTCAGTCTGACTGACAGG

AGACCAGAATGCTGTAGTGATCCTCGCTGTAACTCGAGTCATCCAGA

ACTTTGTGGTGGAAGACGCTGASeq1p:GMRMMFIVFLLVVLATTVVSSTSGRRAFHGRNAAAKASGLVSLTDRR

PECCSDPRCNSSHPELCGGRRSeq2?:PECCSHPACNVDHPEICRSeq2N:ATGGGCATGCGGATGATGTTCACCGTGTTTCTCTTGGTTGTCTTGGC

AACCACCGTCGTTTCCTTCACTTCAGGTCGTCGTACATTTCATGGCA

GGAATGCCGCAGCCAAAGCGTCTGGCCTGGTCAGTCTGACTGACAGG

AGACCAGAATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCAGA

AATTTGTCGTTGAAGACGCTGASeq2P:MGMRMMFTVFLLVVLATTVVSFTSGRRTFRGRNAAAKASFLVSLTDR

RPECCSHPACNVDHPEICRSeq3?:CLDAGEVCDIFFPTCCGYCILLFCASeq3N:ATGAAACTGACGTGTGTGATGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGCCACGGCTGATGACTCCAGCAATGGATTGGGGAATC

TTTTTTTGAAGGCACATCACGAAATGAAGAACCCCGAAGCCTCTAAG

TTGAACGAGAGGTGCCTTGATGCTGGTGAAGTTTGTGATATTTTTTT

TCCAACATGCTGCGGCTATTGCATTCTTCTTTTCTGCGCATAASeq3P:MKLTCVVIVAVLFLTAWTFATADDSSNGLGNLFLKAHHEMKNPEASK

LNERCLDAGEVCDIFFPTCCGYCILLFCASeq4?:YDCEPPGNFCGMIKIGPPCCSGWCFFACASeq4N:ATGAAACTGACGTGCGTGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGTGCCTCACTCCAGCAATGCATTGGAGA

ATCTTTATCTGAAGGCACGTCACGAAATGGAAAACCCTGAAGCCTCT

AAATTGAACACGAGATACGACTGCGAACCTCCTGGAAATTTTTGTGG

CATGATAAAAATTGGGCCGCCTTGCTGCAGTGGCTGGTGCTTTTTCG

CCTGCGCCTAASeq4P:MKLTCVVIVAVLFLTAWTFVTAVPHSSNALENLYLKARHEMENPEAS

KLNTRYDCEPPGNFCGMIKIGPPCCSGWCFFACASeq5?:NYCQEKWDYCPVPFLGSRYCCDGLFCTLFFCASeq5N:ATGAAACTGACGTGCGTGGTGATCGTTGCTGTGCTGTTCTTGACAGC

CTGGACGCTAGTCATGGCTGATGACTCCAACAATGGACTGGCGAATC

TTTTTTCGAAATCACGTGATGAAATGGAGGACCCTGAAGCTGCTAAA

TTGGAGAAAAACTATTGCCAAGAAAAATGGGATTATTGTCCAGTACC

GTTCTTGGGATCGAGGTATTGCTGCGATGGCTTATTCTGTACTCTTT

TCTTCTGCGCTTGASeq5P:MKLTCVVIVAVLFLTAWTLVMADDSNNGLANLFSKSRDEMEDPEAAK

LEKNYCQEKWDYCPVPFLGSRYCCDGLFCTLFFCASeq6?:CYDGGTSCDSGIQCCSGWCIFVCFSeq6N:ATGAAACTGACGTGTGTGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGTGCCTCACTCCAGCAATGCGTTGGAGA

ATCTTTATCTGAAGGCACATCATGAAATGAACAACCCCGAAGCCTCT

GAATTGAACAAGAGGTGCTATGATGGTGGGACAAGTTGCGACTCTGG

AATCCAATGCTGCAGTGGCTGGTGCATTTTCGTCTGCTTCTAASeq6P:MKLTCVVIVAVLFLTAWTFVTAVPHSSNALENLYLKAHHEMNNPEAS

ELNKRCYDGGTSCDSGIQCCSGWCIFVCFSeq7?:CYDSGTSCNTGNQCCSGWCIFVCLSeq7N:ATGAAACTGACGTGCATGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCTGCGCCTCACTCCAGCAATGCGTTGGAGA

ATCTTTATCTGAAGGCACATCATGAAATGAACAACCCCGAAGCCTCT

GAATTGAACAACAGGTGCTATGATAGTGGGACAAGTTGTAACACTGG

AAACCAATGCTGCAGTGGCTGGTGCATTTTCGTCTGCCTCTAASeq7P:MKLTCVVIVAVLFLTAWTFVTAAPHSSNALENLYLKAHHEMNNPEDS

ELNKRCYDSGTSCNTGNQCCSGWCIFVCLSeq8?:CVPYEGPCNWLTQNCCDATCVVFWCLSeq8N:ATGAAACTGACGTGCATGGTGATCGTTGCTGTGCTGTTCTTGACCGC

CTGGACATTCGTCACGGCCATCACCAGCAATGGATTGGAGAATCTTT

TTCCGAATGCACATCACGAAATGAAGAACCCTGAAGCCTCTAAATTG

AACAAGAGGTGCGTTCCATACGAGGGCCCTTGTAATTGGCTTACACA

AAACTGCTGTGATGCAACATGCGTAGTATTTTGGTGCCTATAASeq8P:MKLTCVVIVAVLFLTAWTFVTAITSNGLENLFPNAHHEMKNPEASKL

NKRCVPYEGPCNWLTQNCCDATCVVFWCLSeq9:CRPSGSPCGVTSICCGRCYRGKCT*

* represent that this toxin carboxyl terminal is by amidation Seq9N:ATGAAACTGACGTGCGTGGTGATCGTCGCCGTGCTGCTCCTGACGGC

CTGTCAACTCATCACAGCTGAGGACTCTAGAGGTGCGCAGAAGCATC

GTACCCTGCGGTCGACCGCCAGACGCTCCAAGTCCGAGTTGACTACG

AGATGCAGGCCTAGTGGATCCCCCTGTGGTGTTACTAGTATATGCTG

TGGTAGATGCTATAGGGGTAAATGTACGTAGSeq9P:MKLTCVVIVAVLLLTACQLITAEDSRGAQKHRTLRSTARRSKSELTT

RCRPSGSPCGVTSICCGRCYRGKCTSeq10:CRPSGSPCGVTSICCGRCSRGKCT*

* represent that this toxin carboxyl terminal is by amidation Seq10N:ATGAAACTGACGTGCATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGAGGACTCTAGAGGTACGCAGAAGCA

TCGTACCCTGCGGTCGACCGCCAGACGCTCCAAGTCCGAGTTGACT

ACGAGATGCAGACCTAGTGGATCCCCCTGTGGTGTTACTAGTATAT

GCTGTGGTAGGTGCTCTAGGGGTAAATGTACGTAGSeq10P:MKLTCVVIVAVLLLTACQLITAEDSRGTQKHRTLRSTARRSKSELT

TRCRPSGSPCGVTSICCGRCSRGKCTSeq11?:CKAAGKPCSRIAYNCCTGSCRSGKC*

* represent that this toxin carboxyl terminal is by amidation Seq11N:ATGAAACTGACGTGTATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTACCCTGCGGTCGAAGACCAAACTCTCCATGTCGACTCGCTGC

AAGGCTGCAGGAAAACCATGCAGTAGGATTGCGTATAACTGCTGCA

CCGGTTCTTGCAGATCAGGTAAATGTGGCTGASeq11P:MKLTCVVIVAVLLLTACQLITADDSRGTQKHRTLRSKTKLSMSTRC

KAAGKPCSRIAYNCCTGSCRSGKCGSeq12?:ATDCIEAGNYCGPTVMKICCGFCSPYSKICMNYPKNSeq12N:ATGAAACTGACGTGCATGGTGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTTCCCTGAGGTCGACCACCAAAGTCTCCAAGGCGACTGACTGC

ATTGAAGCCGGAAATTATTGCGGACCTACTGTTATGAAAATCTGCT

GCGGCTTTTGCAGTCCATACAGCAAAATATGTATGAACTATCCCAA

AAATTGASeq12P:MKLTCMVIVAVLLLTACQLITADDSRGTQKHRSLRSTTKVSKATDC

IEAGNYCGPTVMKICCGFCSPYSKICMNYPKNSeq13?:STSCMEAGSYCGSTTRICCGYCAYFGKKCIDYPSNSeq13N:ATGAAACTGACGTGCGTGATGATCGTCGCCGTGCTGCTCCTGACGG

CCTGTCAACTCATCACAGCTGATGACTCCAGAGGTACGCAGAAGCA

TCGTTCCCTGAGGTCGACCACCAAAGTCTCCAAGTCGACTAGCTGC

ATGGAAGCCGGATCTTATTGCGGCTCTACTACGAGAATCTGCTGCG

GTTATTGCGCTTATTTCGGCAAAAAATGTATTGACTATCCCAGCAA

CTGASeq13P:MKLTCVVIVAVLLLTACQLITADDSRGTQKHRSLRSTTKVSKSTSC

MEAGSYCGSTTRICCGYCAYFGKKCIDYPSNSeq14?:DGCSSGGTFCGIHPGLCCSEFCFLWCITFIDSeq14N:ATGAAACTGACGTGTGTGATGATCGTTGCTGTGCTGTTCTTGACCA

CTTGGACATTCGTCACGGCTGATGACTCCAGATATGGATTGAAGAA

TCTTTTTCCGAAGGCACGTCATGAAATGAAGAACCCCGAAGCCTCT

AAATTGAACAAGAGAGATGGGTGCTCTAGTGGTGGTACATTTTGTG

GCATCCATCCAGGACTCTGCTGCAGCGAGTTTTGCTTTCTTTGGTG

CATAACATTTATTGATTGASeq14P:MKLTCVVIVAYLFLTTWTFGTADDSRYGLKNLFPKARHEMKNPEAS

KLNKRDGCSSGGTFCGIHPGLCCSEFCFLWCITFIDB. use: toxin Seq11 has analgesic activities, can be used as a kind of analgesic.