CN1796413A - New T - ultra family conantokins, coded polynucleotide and application - Google Patents

Abstract

This invention describes a new T-CTX, its encoding polynucleotide, the construction units and transformation cells having this encoding polynucleotide, and the reconstruction process for producing the said T-CTX. This invention also describes the artificial synthesis and applications of the said T-CTX.

Description

New T-superfamily conotoxin peptide, its coded polynucleotide and purposes

Technical field

(T-superfamily Conotoxin T-CTX), its coded polynucleotide and purposes, belongs to biological technical field to the present invention relates to new T-superfamily conotoxin peptide.

Background technology

Carnivorous mollusk cone shell (Conus) lives in the tideland of tropical ocean and karang, rock and the sandy end of subtidal zone shoaling water, and minority is distributed in the depth of water and locates for about tens meters to 200 meters, has beautiful shell usually.There are poison gland and sagittate radula in the inside, oral cavity of cone shell, can inject toxin to prey like the radula of syringe and harpoon sample, and its toxicity is very big, and it is dead to cause that the paralysis paralysis appears fainting from fear, tremble and making it in animal.The small peptide animal nerve peptide toxin that this class that we secrete out with cone shell is used to anaesthetize prey be generically and collectively referred to as conotoxin (Conotoxin, CTX).

Cone shell can roughly be divided into ichthyophagy class (pisicvorous), food mollusc (molluscivorous) and food Vermes (vermivorous) three classes according to its feeding habits difference, but the cone shell that has is hunting two classes wherein simultaneously, and polychetes in the sea also usually is their food.Each about 70 kinds of the ichthyophagy in the whole world and food spiral shell cone shells, all the other major parts are carnivorism cone shell (＞350 kinds).And the 100 kinds of different components of having an appointment in every kind of cone shell venom, the same with the secondary metabolite of the alkaloid of plant and microorganism, have of a great variety, baroque characteristic, with also there being allelic variation between a kind of conotoxin of cone shell Different Individual, more increased the diversity of conotoxin.Have at least 50,000 kinds of different conotoxin peptides to have the specific function of regulating various ionic channels and acceptor, thereby formed a series of neurologic agent source, therefore the cone shell venom also is called as " still undeveloped national treasury ".Conotoxin is made up of 6～41 amino-acid residues, is rich in the animal nerve peptide toxin of halfcystine (Cys), and its toxicity is very big, can cause that animal occurs fainting from fear, tremble even benumbing dead.Its chemical structure novelty, biological activity is strong, the selectivity height of target site.The carnivorism conotoxin has lethal effect to multiple worm in addition, can be developed as the polypeptide sterilant, or transforms plant cultivation anti-pest crop new variety.Though it is the thing of nearly more than ten years that conotoxin is carried out profound research, because of conotoxin both can directly be used as natural drug, can be used as the lead compound of medicinal design again.Therefore, conotoxin has leapt to the first place of animal nerve toxin study, becomes the new source of the strong instrument and the new drug development of pharmacology and neuroscience, has caused the extensive attention of whole world scientists.

China's cone shell aboundresources, be the distinctive medicinal marine organisms in China Hainan, the toxin study of most cone shell kinds is not carried out as yet, China more should step up to carry out the research and development of the conotoxin genetic resources with independent intellectual property right, the ocean pharmaceutical industry is had the meaning of particularly important.Content of the present invention adapts to the development trend and the needs of current marine drug conotoxin research just and obtains, and has broad application prospects.

Act on biological intravital different target position according to conotoxin and can be divided into 3 classes: (1) acts on the CTX of voltage gated ion channel, and voltage gated ion channel claims the voltage sensitivity passage again, often with penetrating ion (as Na ⁺, K ⁺, Ca ²⁺Deng) name.(2) act on the CTX of ligand-gated ion channel, comprise nAChR, 5HT3 acceptor, nmda receptor.Ligand gated channel claims chemically-gated channel or mediator dependency passage again, and the latter is by corresponding acceptor name.(3) act on the CTX of other acceptors, CTX is except acting on ionic channel, and also having with G-albumen is target, as: cone shell vassopressin and cone shell inertia element, and 2 kinds of phosphatide (conodipine M and PLA2), they are substantially devoid of disulfide linkage.Acceptor target by its effect can be divided into conotoxin multiple hypotypes such as α, ω, μ, δ.Conservative property according to conotoxin gene and precursor protein signal peptide thereof, conotoxin can be divided into a plurality of superfamilies such as A, O, T, M, P, I and (see Fig. 1, draw TERLAU from HEINRICH, BALDOMERO M.OLIVERA.Conus Venoms:A Rich Source of Novel Ion Channel-Targeted Peptides.Physiol.Rev.2004,84:41-68).Each superfamily can be divided into α, α A, κ A (A-superfamily) again according to acceptor target type, ω, δ, κ, μ O (O-superfamily), μ, ψ, KM family'ies (hypotype) such as (M-superfamilies)

With receptor related process be important step in the life regulate process, the research of receptor protein is identified, and is closely related with the toxin with specific action and high-affinity binding ability.Conotoxin is by combining with the multiple acceptor of N﹠M cytolemma (sodium channel, calcium channel and acetylcholine receptor etc.) specificity, influence and receptor related a series of cell regulate processes, make acceptor research illustrate the molecule mechanism level, become Neuroscience Research and identify the important of acceptor and regulating cell activist mechanism thereof from inferring that analysis level enters

Instrument, and in the treatment of difficult and complicated cases such as Persistent Pain, epilepsy, cardiovascular disease, psychosis, ataxia, spasm, cancer, acquired immune deficiency syndrome (AIDS), neuroprotective, apoplexy, demonstrate tempting application prospect.Multiple conotoxin entered clinical trial (see Table 1, draw slip from LAURA NELSON.Venomous snails:One, and you ' re dead....Nature, 2004,429:798-799).

Table 1 is in the conotoxin of different clinical experimental stages

Voltage gated ion channel (claiming the voltage sensitivity passage again) comprises Ca ²⁺Ionic channel, Na ⁺Ionic channel and K ⁺Ionic channel etc.Ca ²⁺Passage is one of neccessary composition of all excitability cytolemma, is divided into L, T, N, P and Q type, the wherein Ca of N type ²⁺Passage only is present in neuronal tissue's (as vertebrate central nervous system and peripheral nervous system), and it mainly suppresses especially norepinephrine Ca when sympathetic nerve discharges of neurotransmitter ²⁺Enter.T-superfamily conotoxin (CC-CC or CC-CP-C) extensively is present in the various cone shell venom, has 1000 kinds of T-CTX with unique pharmacologically active at least.Studies show that the halfcystine pattern is the T-superfamily conotoxin of CC-CC, as tx5a, its major function is to reduce Ca ²⁺Enter, its molecular target may be Ca ²⁺Ionic channel or influence Ca ²⁺The G albumen pairing acceptor of ionic channel, its concrete molecular target is not identified as yet.The halfcystine pattern is the T-superfamily conotoxin of CC-CP-C, as χ-CTX mr5a, mainly acts on norepinephrine mediator (norepinephrinetransporter).

New T-superfamily conotoxin of the present invention all has its unique receptor-specific and avidity, has value in the research of acceptor, is the drug candidate and the lead drug of new drug development.They are huge at the application potential in Neuroscience Research and new drug development field.The research of the T-superfamily conotoxin that produce in relevant Chinese Hainan does not appear in the newspapers as yet.This demand of the prior art just that the present invention is directed to has great importance to the research and development of marine drug.

Summary of the invention

What the present invention is based on is the marble cone shell (Conus marmoreusLinnaeus), conus (Conus littertus Linnaeus), picture-weaving in silk cone shell (Conustextile Linnaeus), barrel-shaped cone shell (Conus betulinus Linnaeus) and the wart wormwood artemisia cone shell (Conus lividus Hwass) that produce from China Hainan novel T-superfamily conotoxin peptide (T-CTX) with medicinal function of discovery respectively totally 5 kinds.

Therefore, in one aspect, the invention provides new T-superfamily conotoxin peptide (T-CTX).

The present invention provides the polynucleotide of the described peptide of encoding on the other hand.

The present invention also provides the nucleic acid construct that contains described polynucleotide, contains the expression vector of this nucleic acid construct, and the cell that has been transformed by described nucleic acid construct or expression vector.

The present invention also provides the synthetic preparation method of described peptide.

Find among the present invention that described conotoxin peptide has lethal effect to insects such as cockroach, oriental tobacco budworms.Therefore, conotoxin of the present invention can be used as the exploitation of polypeptide sterilant.Its gene can be used for transforming microorganism (for example insect baculovirus, intestinal bacteria and yeast etc.) and plant, develops novel biopesticide, cultivates the anti-pest crop new variety.

Conotoxin peptide of the present invention can play a role by other acceptors such as coupled ion passage and norepinephrine mediator, G albumen pairing acceptors, has analgesic activities.They are the drug candidate and the lead drug of new drug development.

Therefore, the present invention also provides pharmaceutical composition or the insecticides that comprises peptide of the present invention.

Description of drawings

Fig. 1 illustrates conotoxin superfamily and family's (hypotype) classification chart.Wherein shown gene superfamily, disulfide linkage type and known pharmacology target, and the halfcystine pattern of each gene superfamily.Conotoxin is divided into two big classes: a class is the CTX that is rich in disulfide linkage, and another kind of is the CTX that is not rich in disulfide linkage.

Embodiment

In one embodiment, the invention provides new T-superfamily conotoxin peptide (T-CTX), it comprises the aminoacid sequence that is selected from down group:

(1) aminoacid sequence shown in arbitrary among the SEQ ID NO:1-12;

(2) aminoacid sequence shown in arbitrary among the SEQ ID NO:13-24;

(3) aminoacid sequence identical with above-mentioned (1) or (2) described aminoacid sequence at least 80%; Or

(4) because of 1-5, preferred 1-3, more preferably 1-2, most preferably replacement, disappearance, insertion and/or the interpolation and above-mentioned (1) or the different aminoacid sequence of (2) described sequence of 1 amino-acid residue.

Preferably, the aminoacid sequence that described conotoxin peptide has is identical with SEQ ID NO:1-24 about at least 85%, more preferably about at least 90% is identical, and especially preferably about at least 95% is identical, most preferably about at least 97% identical (being called " homeopeptide " in this article).

For the purposes of the present invention, same degree between two or more aminoacid sequences is by BLAST2.0 queries of protein databases program (Aaltschul etc., 1997, nucleic acids research 25:3389-3402) and adopt following parameters to determine: blastall-pblastp-a4-e10-E0-v500-b250-I[inquires about document]-dprot_all, wherein-p refers to program name,-a refers to the server count that will use,-e refers to expected value,-E refers to extend the cost of breach,-v refers to single line description (one-line description) number,-b refers to the ratio logarithm that will show ,-I refers to inquire about document, and-d refers to the database that is used to inquire about.

The aminoacid sequence difference of the aminoacid sequence of homeopeptide and SEQ ID NO:1-24 may be to replace, insert, add and/or lacked 1 or a plurality of, preferred 1-5, more preferably 1-3, especially preferred 1-2,1 amino-acid residue most preferably.Preferably, amino acid change is that character changes less variation, promptly be can the remarkably influenced Protein Folding and/or active conservative amino acid replace; Small segment disappearance, normally 1 to about 5, preferred 1-3, more preferably 1 amino acid whose disappearance; Little amino or C-terminal extend, as the methionine residues of aminoterminal interpolation; The little connection peptides that reaches about 20-25 residue is arranged; Maybe can be by changing little extension such as poly Histidine fragment, epitope or the land that net charge or other function help purifying.

The example that conservative property replaces is the replacement of carrying out in basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and Xie Ansuan), die aromatischen Aminosaeuren (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).The aminoacid replacement that can not change specific activity usually is known in the art, and by for example H.Neurath and R.L.Hill, 1979, at " protein " book, Academic Press described among the New York.Modal replacement is Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly and the replacement that is reversed.

N-end and/or C-end that the present invention also is included in conotoxin peptide of the present invention have merged the fusion polypeptide of other peptide/polypeptide or the fusion polypeptide of cleavable.The technology that produces fusion polypeptide is known in the art, the encoding sequence that comprises the encoding sequence that connects code book invention peptide and described other the peptide/polypeptide of encoding, make them in same frame, and the expression of fusion polypeptide is controlled by identical promotor and terminator.

Polynucleotide

The invention still further relates to the separation polynucleotide of the nucleotide sequence that contains the peptide of the present invention of encoding.In a preferred embodiment, these polynucleotide comprise coding have among the SEQ ID NO:1-24 arbitrary shown in the nucleotide sequence of polypeptide of aminoacid sequence.In a further preferred embodiment, these polynucleotide comprise among nucleotide sequence shown in arbitrary among the SEQ ID NO:25-36 or the SEQ IDNO:25-36 arbitrary shown in corresponding mature polypeptide coding sequence in the sequence.The present invention also comprise coding have among the SEQ ID NO:1-12 arbitrary shown in the nucleotide sequence of polypeptide of aminoacid sequence, different between the mature polypeptide coding sequence shown in arbitrary among it and the SEQ ID NO:25-36 because of the degeneracy of genetic code.Nucleotide sequence of the present invention comprises genome sequence, and corresponding cDNA and RNA sequence.Term used herein " nucleotide sequence " is understood to include synthetic DNA in all interior these class mutation.

The invention still further relates to the active conotoxin peptide of coding, with SEQ ID NO:25-36 in the mature polypeptide coding sequence shown in arbitrary the nucleotide sequence of certain homology is arranged, the homology degree is at least about 70%, preferred about 80%, more preferably from about 90%, also will be preferably about 95%, most preferably about 97% homology.With regard to the object of the invention, the homology degree between two nucleotide sequences is by Wilbur-Lipman method (Wilbur ﹠amp; Lipman, 1983, the journal 80:726-730 of NAS) uses LASERGENE ^TMMEGALIGN ^TMSoftware (DNASTAR, Inc., Madison, WI) and homogeny table and following multiple reduced parameter: breach point penalty and notch length point penalty are 10 and determine.Reduced parameter is Ktuple=3 in pairs, breach point penalty=3, window=20.

During the similar substantially polypeptide of synthetic and polypeptide of the present invention, have necessity the nucleotide sequence of code book invention polypeptide is modified.Term and described polypeptide " similar substantially " are meant the polypeptide of non-natural form.These polypeptide may be different on some processing mode with the polypeptide that is separated to from natural origin.For example, may wish to synthesize variant polypeptides with for example site-directed mutagenesis, these variants have different thermostabilitys or optimal pH etc.Can on the nucleotide sequence basis of the mature peptide encoding part of SEQ ID NO:25-36, construct similar sequence; And/or replace and make up by introducing Nucleotide, described replacement can not make the aminoacid sequence of generation different with former nucleotide sequence encoded polypeptides, but they meet enzyme and prepare the use habit of used host living beings to codon; Or replace by the Nucleotide that introducing can produce the different aminoacids sequence and to make up.The summary that replaces about Nucleotide, referring to for example Ford etc., 1991, protein expression and purifying 2:95-107.

The invention still further relates to comprise the active conotoxin peptide of coding, with SEQ ID NO:25-36 in the mature polypeptide coding sequence shown in arbitrary or the polynucleotide of the interfertile nucleotide sequence of its complementary sequence.About the hybridization between the polynucleotide, there is numerous documents can be for reference in the prior art, comprise for example Sambrook etc., molecular cloning laboratory manual, second edition, cold spring harbor laboratory, cold spring port, 1989.Can use the rigorous condition of various degree in the hybridization, for example moderate, moderate-highly, perhaps highly rigorous condition.Rigorous more condition, the complementary degree that forms the duplex requirement is high more.Can pass through the rigorous degree of temperature, concentration and probe concentration, probe length, ionic strength, time or the like control.For the double-stranded DNA gene probe, hybridize in being lower than DNA heterozygote melting temperature (Tm) [melting temperature, Tm]) in 6X SSPE, 5X DenhardtShi solution, 0.1%SDS, 0.1mg/ml denatured DNA, spend the night under the 20-25 ℃.Clean following carrying out usually: in Tm-20 ℃ in 0.2X SSPE, 0.1%SDS one time 15 minutes (the rigorous condition of moderate is cleaned).

For oligonucleotide probe, hybridize in 6X SSPE, 5X DenhardtShi solution, 0.1%SDS, 0.1mg/ml denatured DNA, spending the night under the melting temperature (Tm) Tm 10-20 that is lower than heterozygote ℃.Clean following carrying out usually: in hybridization temperature one time 15 minutes (the rigorous condition of moderate is cleaned) in 1X SSPE, 0.1%SDS

Nucleic acid construct

The invention still further relates to the nucleic acid construct of 1 or a plurality of regulating and controlling sequences that comprise nucleotide sequence of the present invention and can be operatively connected with it, described regulating and controlling sequence can instruct encoding sequence to express in proper host cell under its consistency condition.Expression be understood to include polypeptide produce in related any step, include, but are not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.

" nucleic acid construct " is defined as strand or double chain acid molecule in the text, and they separate from natural gene, and be perhaps modified and contain in the non-natural mode and make up and nucleic acid fragment arranged side by side.When nucleic acid construct comprises when expressing essential all regulating and controlling sequences of encoding sequence of the present invention term nucleic acid construct and expression cassette synonym.Term " encoding sequence " is defined as the part of directly determining the aminoacid sequence of its protein product in the nucleotide sequence in the text.The border of encoding sequence is normally determined by the ribosome bind site (for prokaryotic cell prokaryocyte) of next-door neighbour mRNA 5 ' end opening code-reading frame upstream and the transcription termination sequence in next-door neighbour mRNA 3 ' end opening code-reading frame downstream.Encoding sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Can operate the isolated nucleic acid sequences of coding peptide of the present invention in many ways, make it express described peptide.May expect or must before inserting carrier, process that this depends on expression vector to nucleotide sequence.The technology of using recombinant DNA method modification of nucleic acids sequence is known in the art.

Herein term " control sequence " be defined as comprise express peptide of the present invention institute must or favourable all components.Each regulating and controlling sequence can be natural that contain or external for the nucleic acid encoding sequence.These regulating and controlling sequences include, but not limited to leader sequence, polyadenylic acid sequence, propeptide sequence, promotor, signal sequence and transcription terminator.Bottom line, regulating and controlling sequence will comprise promotor and the termination signal of transcribing and translating.So that the coding region of regulating and controlling sequence with the nucleic acid encoding sequence is connected, can provide the regulating and controlling sequence of belt lacing in order to import specific restriction site.Term " can be operatively connected " and be defined as a kind of like this conformation in the text, and wherein regulating and controlling sequence is positioned at the appropriate location of the encoding sequence of relative dna sequence dna, so that regulating and controlling sequence instructs polypeptide expression.

Regulating and controlling sequence can be suitable promoter sequence, can be by the nucleotide sequence of the host cell of express nucleic acid sequence identification.Promoter sequence contains the transcription regulating nucleotide sequence that mediates expression of polypeptides.Promotor can be any nucleotide sequence that transcriptional activity is arranged in selected host cell, comprises sudden change, brachymemma and promotor heterozygosis, can get the gene of polypeptide in the outer or born of the same parents of own coding and host cell homology or allogenic born of the same parents.

Regulating and controlling sequence can also be suitable transcription termination sequence, thereby can be discerned one section sequence that termination is transcribed by host cell.Terminator sequence can be operatively connected 3 ' end in the nucleic acid encoding sequence.Any terminator that can bring into play function in selected host cell may be used to the present invention.

Regulating and controlling sequence can also be suitable leader sequence, promptly to the crucial mRNA non-translational region of the translation of host cell.Leader sequence can be operatively connected 5 ' end in the nucleic acid encoding sequence.Any leader sequence that can bring into play function in selected host cell all can be used for the present invention.

Regulating and controlling sequence can also be a signal peptide coding region, and this district's coding is connected in the N-terminal aminoacid sequence of polypeptide for one section, can guide coded polypeptide to enter the emiocytosis approach.5 ' the end in nucleic acid sequence encoding district may be natural contain the signal peptide coding region that the translation frame as one man is connected naturally with the coding region fragment of secrete polypeptide.Perhaps, can to contain encoding sequence be external signal peptide coding region to 5 ' of coding region end.When encoding sequence does not under normal circumstances contain signal peptide coding region, may need to add the extraneous signal peptide-coding region.Perhaps, can replace the natural signals peptide-coding region simply to strengthen the polypeptide secretion with external signal peptide coding region.But the signal peptide coding region that the polypeptide after any energy guiding is expressed enters the Secretory Pathway of used host cell may be used to the present invention.

Regulating and controlling sequence can also be peptide original encoding district, and this district's coding is positioned at the aminoterminal one section aminoacid sequence of polypeptide.The gained polypeptide is called as proenzyme or propolypeptide.Propolypeptide does not have activity usually, can be by catalysis or self-catalysis and be converted into sophisticated active polypeptide from propolypeptide cutting peptide is former.

When the N-terminal of polypeptide promptly has signal peptide that the former district of peptide is arranged again, the N-terminal of peptide former district next-door neighbour polypeptide, the signal peptide district then is close to the N-terminal in the former district of peptide.

The regulating and controlling sequence that interpolation can be regulated expression of polypeptides according to the growing state of host cell may also be needs.The example of regulator control system is that those can be reacted to chemistry or physical stimulation thing (being included under the situation of regulating compound), thereby opens or close the system of genetic expression.Other examples of regulating and controlling sequence are those regulating and controlling sequences that can make gene amplification.In these examples, nucleic acid encoding sequence and regulating and controlling sequence can should be operatively connected together.

Expression vector

The invention still further relates to and comprise nucleotide sequence of the present invention, promotor and transcribe and the recombinant expression vector of translation termination signal.Above-mentioned various nucleic acid and regulating and controlling sequence can be linked together prepares recombinant expression vector, and this carrier can comprise 1 or a plurality of restriction site easily, so that insert in these sites or replace the nucleic acid encoding sequence.Perhaps, can express nucleotide sequence of the present invention by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted suitable expression vector.Preparation is during expression vector, can make encoding sequence be arranged in carrier so that can be operatively connected with suitable expression regulation sequence.

Recombinant expression vector can be any carrier (for example plasmid or virus) of being convenient to carry out recombinant DNA operation and express nucleic acid sequence.The consistency of the host cell that carrier and it will import is depended in the selection of carrier usually.Carrier can be linearity or closed loop plasmid.

Carrier can be self-replicating type carrier (promptly be present in extrachromosomal complete structure, can be independent of karyomit(e) and duplicate), for example plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can comprise any mechanism that guarantees self-replacation.Perhaps, carrier be one when importing host cell, with the carrier that is incorporated in the genome and duplicates with the karyomit(e) that is incorporated into.In addition, can use single carrier or plasmid, or totally comprise and will import the two or more carriers or the plasmid of all DNA of host cell gene group, or transposon.

Preferred carrier of the present invention contains 1 or a plurality of selective marker of being convenient to select transformant.Selective marker is such gene, and its product is given to the resistance of biocide or virus, to the resistance of heavy metal, or gives auxotroph prototroph etc.The dal gene of the example of bacterium selective marker such as subtilis or Bacillus licheniformis, the perhaps resistance marker of microbiotic such as penbritin, kantlex, paraxin or tsiklomitsin.

Preferred carrier of the present invention comprises can make the carrier stable integration in the host cell gene group, or guarantees carrier to be independent of cellular genome in cell and carry out the element of self-replicating.

With regard to the situation of carrying out self-replicating, carrier can also comprise replication orgin, and carrier can independently be duplicated in target host cell.Replication orgin can have makes its sudden change that becomes responsive to temperature type in host cell (referring to for example, fEhrlich, 1978, the journal 75:1433 of NAS).

Can insert the nucleotide sequence of the present invention of copy more than 1 to improve the output of this gene product to host cell.The copy number increase of this nucleotide sequence can be by inserting 1 additional copies of this sequence in the host cell gene group at least, perhaps insert a selective marker that can increase with this nucleotide sequence, by culturing cell in the presence of the suitable selective reagents is being arranged, thereby pick out the cell that selected marker that containing the amplification copy is contained the additional copies nucleotide sequence.

It is well-known to those skilled in the art (referring to for example Sambrook etc., molecular cloning laboratory manual, second edition being used to connect the operation that above-mentioned each element makes up recombinant expression vector of the present invention, press of cold spring harbor laboratory, the cold spring port, New York, 1989).

Host cell

The invention still further relates to the recombinant host cell that comprises the nucleotide sequence of the present invention that can be used to the recombinant production polypeptide.The carrier that comprises the present invention's nucleotide sequence can be imported host cell, thereby this carrier is maintained with the outer carrier format of the karyomit(e) of above-mentioned chromosomal integration body or self-replacation.Term " host cell " is contained the sudden change that takes place between any because replicative phase and the offspring different with parental cell.Peptide coding gene and source thereof are depended in the selection of host cell to a great extent.

Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, for example bacterium or yeast cell.Can carrier be imported host cell by technology well known to those skilled in the art.

The preparation method

The invention still further relates to recombinates prepares the method for peptide of the present invention, and this method comprises: (a) be suitable for producing under the condition of described peptide, cultivating the host cell that contains nucleic acid construct, this nucleic acid construct comprises the nucleotide sequence of the described peptide of encoding; (b) reclaim this peptide.

In preparation method of the present invention, with means known in the art culturing cell in the nutritional medium that suitable polypeptide produces.For example, can be in suitable medium, allowing under expression of polypeptides and/or the isolating condition, come culturing cell by shake-flask culture, laboratory or industrial fermentation jar middle and small scale or large scale fermentation (comprise continuously, in batches, batch charging or solid state fermentation).In the suitable medium that comprises carbon and nitrogenous source and inorganic salt, adopt step known in the art to cultivate.Suitable medium can be provided or can be prepared with reference to disclosed the composition (for example, described in the catalogue of American type culture collection) by supplier.If polypeptide is secreted in the substratum, then can directly from substratum, reclaim polypeptide.If polypeptide is not secreted, can from cell lysate, reclaim.

Can reclaim the polypeptide that is produced with means known in the art.For example, can from substratum, reclaim polypeptide by routine operation (including, but are not limited to centrifugal, filtration, extracting, spraying drying, evaporation or precipitation).

Can come purifying polypeptide of the present invention by various operations known in the art, these operations comprise, but (for example be not limited to chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography), electrophoresis (for example, preparation property isoelectric focusing), differential solubleness (for example ammonium sulfate precipitation), SDS-PAGE or extracting (referring to for example, protein purification, J.C.Janson and Lars Ryden compile, VCH Publishers, New York, 1989).

Transgenic animal and plant

The invention still further relates to the animal or plant cell that has transformed nucleotide sequence of the present invention, vegetable cells such as preferably wheat, corn, paddy rice, soybean are given and are transformed the new proterties (as insect-resistance) of host.This can be by technology well known to those skilled in the art, with construct transformed animal disclosed herein or vegetable cell and realize.

The method and formulation that is used for Pest Control

Can use conotoxin peptide of the present invention or polynucleotide to realize Pest Control by the several different methods that those skilled in the art will know that.These methods comprise for example recombinant microorganism is applied to insect (or their location) and with the coding conotoxin peptide of the present invention gene-transformed plant.Conversion can use routine techniques to carry out by those skilled in the art.The necessary material that is used for these conversions disclosed herein, perhaps those skilled in the art can be easy to obtain by other method.

The preparation that contains conotoxin peptide or comprise the recombinant microorganism of polynucleotide of the present invention of preparation can be applied to soil.The product of preparation can also be covered material or root processing or the whole plant in late period in plant growth cycle as seed and handle application.Preparation can comprise diffusion-thickening adjuvant, stablizer, other insecticidal additive or tensio-active agent.That liquid preparation can be based on water or non-water, and use with foam, gel, suspension, emulsifiable concentrate or the like form.Composition can comprise rheological agent, tensio-active agent, emulsifying agent, dispersion agent or polymkeric substance.

It will be understood by those skilled in the art that insecticide concentration will extensively change owing to the person's character of special preparation, particularly can be used as enriched material or directly use.Sterilant will exist at least 1% (weight), and may be 100% (weight meter).Drying agent has the sterilant of about 1-95% (weight meter) usually, and liquid preparation will be normally the about 1-60% of solid weight in the liquid phase.The preparation that contains cell will contain about 10 usually ²-about 10 ⁴Individual cell/mg.These preparations will use with the about 50mg of per hectare (liquid or do)-1kg or more amount.By spray, spread, spill, or the like, preparation can be applied to insect environment, for example soil and plant.

Pharmaceutical composition

The invention still further relates to the pharmaceutical composition that contains peptide of the present invention and pharmaceutical acceptable carrier and/or vehicle.Described pharmaceutical composition can be used for alleviation or treatment and ischemia injury diseases associated or illness.In one embodiment, the pharmaceutical composition that contains the peptide of the present invention for the treatment of significant quantity is beneficial to medicinal mode and prepares and administration, and need consider individual patient clinical condition, transport site, medication, administration schedule and the known other factors of doctor.Therefore be used for " significant quantity " of this paper purpose consideration decision by these aspects.

The pharmaceutical composition that contains the polypeptide of the present invention for the treatment of significant quantity can be oral, administration etc. in the parenterai administration, brain pond." pharmaceutical acceptable carrier " refers to the prescription subsidiary of nontoxic solid, semisolid or liquid filler material, diluent, capsule material or any kind.The administering mode of term used herein " parenteral " expression comprises intravenously, intramuscular, intraperitoneal, breastbone is interior, subcutaneous and intra-articular injection and infusion.Polypeptide of the present invention also can pass through slow-released system administration rightly.

Also can use conotoxin peptide of the present invention or gene as useful probe, study and identify that the animal voltage gated ion channel (comprises Ca ²⁺, Na ⁺And K ⁺Ionic channel etc.) and the important tool of acceptor such as ligand-gated ion channel and regulating cell activist mechanism thereof; Determine the different subtype of ionic channel as molecular probe; As molecular model, the design new drug; As the polypeptide sterilant, be developed as novel biopesticide etc.In the research of neuroscience acceptor, in the treatment of difficult and complicated cases such as Persistent Pain, epilepsy, cardiovascular disease, psychosis, ataxia, spasm, cancer, acquired immune deficiency syndrome (AIDS), neuroprotective, apoplexy, have broad application prospects

The invention will be further described below with reference to embodiment.Provide just explanation for example of these embodiment, the scope that they do not limit the present invention in any way.

Embodiment

The clone of embodiment 1 T-superfamily conotoxin gene

The extraction of the total RNA of 1 cone shell

With from marble cone shell (Conus marmoreusLinnaeus), conus (Conus littertus Linnaeus), picture-weaving in silk cone shell (Conustextile Linnaeus), barrel-shaped cone shell (Conus betulinus Linnaeus) and the wart wormwood artemisia cone shell (Conus lividus Hwass) of coastal collections such as Hainan Island, the Xisha Islands cone shell live body of totally 5 kinds be material.With the total RNA extraction agent of the centrifugal tissue/cell of a small amount of post box (Shanghai China Shun biotechnology company limited), or, extract total RNA by operational manual with Total RNA Isolation System test kit (Promega).The RNA extraction agent box that produces with Shanghai China Shun is an example below.

Cone shell poison gland or malicious effective liquid nitrogen freezing and grinding powder, change in the 1.5mL centrifuge tube earlier.Add 500 μ L TCL liquid (RNA lysates in the sample; Shanghai Hua Shun); centrifugal 3 minutes of 12000rpm behind the mixing → shift supernatant also adds in supernatant liquor in the thorough mixing of ethanol → immigration RNA adsorption column of 250 μ L 75%; centrifugal 30 seconds of 10000rpm; outwell the liquid in the collection tube; adsorption column is moved in the same collection tube → adds 500 μ L RP liquid (protein liquid removal); centrifugal 30 seconds of 10000rpm; outwell the liquid in the collection tube; adsorption column is moved in the same collection tube → adds 500 μ L W3 liquid (washings); centrifugal 15 seconds of 10000rpm; outwell the liquid in the collection tube; adsorption column is moved in the same collection tube → with method with W3 liquid washed twice → 10000rpm centrifugal 1 minute → adsorption column is moved in the clean 1.5mL centrifuge tube of another one → add 50ul pure water dissolving RNA in adsorption film central authorities, room temperature was put 1 minute → 10000rpm only centrifugal 1 minute → the 1.5mL centrifuge tube is put-70 ℃ of preservations.

2 cDNA's is synthetic

The total RNA that extracts with step 1 is a template, is primer with Oligo (dT) 15 and 3 '-RACEAdapter Primer respectively, adopts two kinds of methods to synthesize cDNA.Division is as follows.

Method 1, the synthetic cDNA of employing ThermoScriptTM RNase H-Transcriptase test kit (Invitrogen).

Reacted constituent: 3 '-RACE primer (5 ' GGC CAC GCG TCG ACT AGT AC (T) 17-3 '), 0.5 μ g, total RNA 2 μ g, 1mmol.1-1 dNTP mixture, redistilled water, 5 * cDNA synthesizes damping fluid, 0.1mol.1-1 DTT, RNaseOUTTM RNAsin (RNaseOUTTM Recombinant Ribonuclease Inhibitor), ThermoScriptTMRT 20U.The reaction cumulative volume is 20 μ l.Response procedures: earlier total RNA template is bathed 5min 65 ℃ of temperature, add other compositions again; Reaction solution is in 50 ℃ of incubation 60min, 85 ℃ of incubation 5min termination reactions; Reverse transcription product is put-20 ℃ of preservations.

Method 2, the synthetic cDNA of employing AMV Transcriptase test kit (Invitrogen).

Reacted constituent: the total RNA of 0.5 μ g Oligo (dT), 15,2 μ g, reverse transcription damping fluid, 1mmol.1-1 dNTP, 20U recombinant RNA enzyme inhibitors ribonuclease inhibitor (RecombinantRnasin Ribonuclease Inbibitor), 25U AMV ThermoScript II, redistilled water.The reaction cumulative volume is 20 μ l.Response procedures: elder generation at 70 ℃ of incubation 10min, adds other reacted constituents with total RNA again; In 42 ℃ of incubation 1h, 95 ℃ of termination reaction 5min; Reverse transcription product is put-20 ℃ of preservations.

Get 5 μ l reverse transcription products and carry out the detection of 1% agarose electrophoresis.

3 reverse transcription PCRs reactions (RT-PCR) and 3-RACE amplification

With step 2 synthetic cDNA is template, according to T-superfamily conotoxin signal peptide zone conserved regions and 3 ' non-translational region sequence (Craig S.Walker, Douglas Steel et al.The T-superfamily of Conotoxins. Biol Chem, 1999,274 (43): 30664-30671.J.Michael McIntosh, Gloria O.Corpuz, et al.Isolation and Characterization of a Novel Conus Peptide withApparent Antinociceptive Activity.J.Biol.Chem., 2000,275 (42): 32391-32397.), design T-superfamily CTX primer, totally 4 upstream primers, article 3, downstream primer (primer pairing see below table 3) and 3-RACE joint primer (5 ' GGC CAC GCGTCG ACT AGT AC 3 ') carry out PCR respectively and react.PCR reaction cumulative volume is 25ul, comprising: cDNA template 50ng, 10 * PCR buffer, 200uM dNTPs, 1umol upstream primer and downstream primer or 3 '-RACE joint primer (5 ' GGC CAC GCG TCG ACT AGT AC3 '), 2mM MgCl2,1U Taq enzyme and deionized water.Cycling program: 94 ℃ of pre-sex change 3 minutes, 94 ℃ of sex change 30 seconds, 50 ℃ (or 55 ℃) annealing 30 seconds, 72 ℃ were extended 30 seconds, and after 35 circulations, 72 ℃ were extended 2 minutes again.Pcr amplification product is got the 5ul point sample, detects with 1.5% agarose gel electrophoresis.

The clone of 4 PCR products and order-checking

Reclaim above-mentioned specific PCR product, be connected back transformed into escherichia coli XL1 bacterial strain (preservation of contriver laboratory) with T-easy carrier (Promega), utilize blue white bacterium colony and amicillin resistance to select recon, extracting and purifying recon plasmid is used for sequencing analysis.Through sequential analysis relatively, 12 kinds of novel T-superfamily conotoxin mature peptides of the present invention (T-CTX, SEQ IDNO:1-12) have been obtained.Mature peptide is to infer according to its propetide (SEQ ID NO:13-24) and conotoxin characteristics, and propetide is to infer according to precursor-gene (SEQ ID NO:25-36).Situations such as T-CTX numbering and source cone shell kind thereof are listed in table 2.

12 kinds of T-superfamily conotoxin mature peptides (T-CTX, SEQ ID NO:1-12) sequence sees Table 3.12 kinds of T-CTX propetides (T-CTX, SEQ ID NO:13-24) sequence sees Table 4.The coded polynucleotide of 12 kinds of T-CTX (DNA) sequence (SEQ ID NO:25-36) sees Table 5.

Table 2 T-superfamily conotoxin peptide (SEQ ID NO:1-12), propetide (SEQ ID NO:13-24) and coded polynucleotide (SEQ ID NO:25-36) numbering and source

SEQ ID NO	The mature peptide title	SEQ ID NO	The propetide title	SEQ ID NO	The precursor-gene title	Source cone shell kind	Used T-superfamily CTX primer Δ
								1	TeA31M	13	TeA31P	25	TeA31N	Picture-weaving in silk cone shell C. textile	Upstream primer (20bp) 5 ' GAGGAAGCTGACTACAAGCA, 3 ' downstream primer (20bp) 5 ' GCTTTAGGTC ATCCAGTTCC 3 '
2	BeB34M	14	BeB34P	26	BeB34N	Barrel-shaped cone shell C. betulinus
							3	LiC32M	15	LiC32P	27	LiC32N	Wart wormwood artemisia cone shell C. lividus
4	LiC33M	16	LiC33P	28	LiC33N
						5	LeD33M	17	LeD33P	29	LeD33N	Conus C. littertus
6	TeAr151M	18	TeAr151 P	30	TeAr15 1N								Picture-weaving in silk cone shell C. textile	Upstream primer (20bp) 5 ' the TGATCCCTGCAC GGCGAATC 3 ' of SEQ ID NO:6-7
						7	TeAr154M	19	TeAr154 P	31	TeAr15 4N
8	TeAr193M	20	TeAr193 P	32	TeAr19 3N							Picture-weaving in silk cone shell C. textile			Upstream primer (20bp) 5 ' the CCTGCAGGTAC TCAACGAA 3 ' of SEQ ID NO:8-9
						9	LeDr192M	21	LeDr192 P	33	LeDr19 2N		Conus C. littertus
10	LeDr243M	22	LeDr243 P	34	LeDr24 3N							Conus C. littertus		Upstream primer (20bp) 5 ' GGAAGCTGACTACAAGCAGA3 '
						11	LiC121M	23	LiC121P	35	LiC121N		Wart wormwood artemisia cone shell C. lividus		Upstream primer (20bp) 5 ' GGAAGCTGACTACAAGCAGA3 ' downstream primer (20bp) 5 ' CAAATGATGTA ATTACTGAC3 '
12	MaI126M	24	MaI126P	36	MaI126N							Marble cone shell C. marmoreus

Δ is annotated: the downstream primer of SEQ ID NO:30-34 is 3 '-RACE adapter (20bp), 5 '-GGCCACGCGTCGACTAGTAC-3 '

The sequence of table 3 T-superfamily conotoxin peptide (T-CTX, SEQ ID NO:1-12)

SEQ ID NO	The mature peptide title	Total number of atnino acid	The T-CTX sequence, direction: N ' → C ', # represent this toxin carboxyl terminal by amidation
				1	TeA31M	11	ICCYPNVWCCD
2	BeB34M	11	ACCPYEPSCCI
				3	LiC32M	15	LWQNTWCCRDHLRCC#
4	LiC33M	14	ALCCYGYRFCCPIF#
				5	LeD33M	12	DCCPAKMFCCQW
6	TeAr151M	11	VCCRPMQDCCS#
				7	TeAr154M	12	VNCCPIDESCCS
8	TeAr193M	9	NCCRRQICC#
				9	LeDr192M	15	ECCEDGWCCTAAPLT#
10	LeDr243M	11	PCCSIHDNSCC#
				11	LiC121M	15	ALCCYGYRFCCPNFR
12	MaI126M	13	NGVCCGYKLCHPC

12 kinds of T-superfamily conotoxins of table 4 propeptide sequence (SEQ ID NO:13-24)

SEQ ID NO	The propetide title	Total number of atnino acid	The T-CTX propeptide sequence, direction: N ' → C '
				13	TeA31P	61	MRCLPVFVILLLLIASVPSDAVQLKTKDDMPLPSFNGNAR RTPRMLSNKR ICCYPNVWCCD
14	BeB34P	61	MRGLPVFVILLLLIASEPSVDARPKTKADVPLTSLNDNAK RTLQILRNKR ACCPYEPSCCI
				15	LiC32P	61	MRCVPVFIILLLLSPSAPSVDAHPKTKDDVPLASFHDDAK RTLQRLWQNTWCCRDHLRCCG
16	LiC33P	67	MRCVPVFIILLLLSPSAPSVDAHPKTKDDVPLASFHDDAK RTLQRLWIKALCCYGYRFCCPIFGKGK
				17	LeD33P	62	MRFLPVFIILLLLIPSAPSVDAQPMTKDDVPLSSLHDNAK RALQMFWNKRDCCPAKMFCCQW
18	TeAr151P	62	MRCLPVFVVLLLLIASAPSVDAQPKTKDDVPLAPLHDNIQ NTLQTLRKKVCCRPMQDCCSGK
				19	TeAr154P	61	MHCLPVFVILLLLTASGLSVDARPKTEDDVPLSSFRDNTK STLQRLLKRVNCCPIDESCCS
20	TeAr193P	63	MRCLPVFVILLLLIASAPSVDAQPKTKDDIPQASFLDNAK RYLQVLESKRNCCRRQICCGRTK
				21	LeDr192P	67	MRCFPVFIILLLLIASAPCFDARTKTDDDVPLSPLRDNLK RTIRTRLNIRECCEDGWCCTAAPLTGR
22	LeDr243P	63	MRCLPVFVILLLLIASTPSIDARPKTKDDMPLASFNDNAK RILQILSRKPCCSIHDNSCCGLG
				23	LiC121P	64	MRCVPVFIILLLLSPSAPSVDAHPKTKDDVPLASFHDDAK RTLQRLWIKALCCYGYRFCCPNFR
24	MaI126P	61	MRCLPVLIILLLLTASAPGVVVLPKTEDDVPMSSVYGNGK SILRGILRNGVCCGYKLCHPC

Table 5 T-CTX coded polynucleotide (DNA) sequence (SEQ ID NO:25-36)

SEQ ID NO	The precursor-gene title	Base is counted bp	T-CTX coded polynucleotide (DNA) sequence (precursor-gene sequence), direction: 5 ' → 3 '
				25	TeA31N	389	GAGGAAGCTGACTACAAGCAGAATGCGCTGCCTACCAGTCTTCGTC ATTCTTCTGCTGCTGATTGCATCTGTACCTAGCGATGCTGTCCAAC TGAAGACCAAAGATGATATGCCCCTGCCATCTTTCAACGGTAATGC AAGACGAACCCCGCGAATGCTTTCAAACAAACGCATTTGCTGCTAC CCCAACGTTTGGTGCTGTGATTGATGATAAAGGAACATGACTTTGG ATGAGACCCCTGCAAACTGTCCCTGGATGTGAGATTTGGAAAGCAG ACTGTTCCTTTCGCACGTATTCGTGAAATTTCGAATGGTCGTTAAC AACACGCTGCCACTTGCAAGCTACTATCTCTCTGTCCTTTCATCTG CGGAACTGGATGACCTAAAGC
26	BeB34N	392	GAGGAAGCTGACTACAAGCAGAATGCGCGGTCTCCCAGTCTTCGTC ATTCTTCTGCTGCTGATTGCATCTGAACCTAGCGTTGATGCCCGAC CGAAGACTAAAGCTGATGTGCCCCTGACATCTCTCAACGATAATGC AAAGCGAACCCTACAAATACTTCGGAACAAACGCGCTTGCTGCCCG TACGAACCTTCGTGCTGTATTTAACATGATGAAAAGAAACGACTTT GGATGAGACCCCTGCAAACTGTCCCTGGATGTGAAATTTGGAAAGC AGACTGTTCCTTTCGCACGTGTTCGTGGAATTTCGAATGGTCGTTA ACAACACACTGCCACGTGCGAGCTACTATCTCTCTGTCCTTTTATC TGTGGAACTGGATGACCTAAAGCA

27	LiC32N	471	GAGGAAGCTGACTACAAGCAGAATGCGCTGTGTCCCAGTCTTCATC ATTCTTCTGCTGCTGAGTCCATCTGCACCTAGCGTTGATGCCCATC CGAAGACCAAAGATGATGTGCCCCTGGCATCATTCCATGATGATGC AAAGCGAACCCTACAAAGACTTTGGCAAAATACCTGGTGCTGCAGA GATCATCTTCGGTGCTGTGGCTGACCAAGCTGATGCATGCATGGAA CGTCTGGTGAACCTTTGGACAGGGAATGTGGAAGACATCTTGATTG CTGGCGCTATGGATGATAAAGGAAAATGATTTTTGATGAGACCCCT GCGAACTGTCCCTGGATGTGAAATTTGGACTGCAGACTGTTCCTTT CGCACGTGTTCGTGGAATTTCGAATGGTCGTTAACAACACGCTGCC ACTTGCAAGCTATTATCTCTCTGTCCCTTTATCTGTGGAACTGGAT GACCTAAAGCA
				28	LiC33N	392	GAGGAAGCTGACTACAAGCAGAATGCGCTGTGTCCCAGTCTTCATC ATTCTTCTGCTGCTGAGTCCATCTGCACCTAGCGTTGATGCCCATC CGAAGACCAAAGATGATGTGCCCCTGGCATCATTCCATGATGATGC AAAGCGAACCCTACAACGACTTTGGATAAAAGCCTTGTGCTGCTAC GGATATAGATTTTGTTGCCCCATTTTCGGTAAAGGAAAATGATTTT GAATGAGACCCCTGCGAACTGTCCCTGGATGTGAAATTTGGACTGC AGACTGTTCCTTTCGCACGTGTTCGTGGAATTTCGAATGGTCGTTA ACAACACGCTGCCACTTGCAAGCTATTATCTCTCTGTCCCTTTATC TGTGGAACTGGATGACCTAAAGCA
29	LeD33N	384	GAGGAAGCTGACTACAAGCAGAATGCGCTTTCTCCCAGTCTTCATC ATTCTTCTGCTGCTGATTCCATCTGCACCCAGCGTTGATGCACAAC CGATGACCAAAGATGATGTGCCCCTGTCATCTCTCCATGATAATGC AAAGCGAGCCCTACAAATGTTTTGGAACAAACGCGATTGCTGCCCA GCAAAAATGTTCTGCTGCCAATGGTGAAGGGAAATGACTTTGGATG AGACCCCTGCAATGTCCCTGGATGTGAGATTTGGAGAGCAGACTGT TCCTTTCGCGCGTGTTCGTGGAATTTCGAATGGTCGTTAACAACAT GCTGCCACTTGCAAGCTACTATCTCTTTGTCCTTTCATCTGTGGAA CTGGATGACCTAAAGC

30

TeAr151 N

843

TTGATCCCTGCACGGCGAATCCACCTGGCAGGTACTCAACGAACTT CAAGACACATTCTGTTCACCTGGACACGGGAAGCTGACTACAGGCA GAATGCGCTGTCTCCCAGTCTTCGTCGTTCTTCTGCTGCTGATTGC ATCTGCACCTAGCGTTGATGCCCAACCGAAGACCAAAGATGATGTG CCCCTGGCACCTTTGCACGATAATATACAGAATACTCTACAAACAC TTCGGAAGAAAGTCTGCTGCCGCCCGATGCAGGATTGCTGTTCAGG GAAATGAAGGGAAATGAATTTGGATGAGACCCCTGCGAACTGTCCC TGGATGTGAGATTTGGAAAGCAGACTGTTCCTTTCGCACGTGTTCG TGGAATTTCGAATGGTCGTTAACAACACCCTGCCACTTGCAAGCTA CTATCTCTCTGTCCTTTCATCTGCGGAACTGTATGATCTAATAACT GAAATGTCATAGACATTATTAAATGGGTATACACTATGACCATGTA ACCTGTAATTACATCATTTGGACCTTTTGAAATATTTTTCAAAATG TTAAGATTTTTCCCCCTGGAAAGGTCTTTTGAAGTTAAATATTTTA GTATGTTGTTTTGCATACAAGTTATAGAATGCTGTCTTTCTTTTTG TTCCCACATCAATGGTGGGGGCAGAAATTATTTGTTTTGATCATTG TAATTATGACCTGCATTTAGTGCTATAGTGATTGCATTTTCAGCGT TGAATGTTTTATCTGCAAACAGAAAGTGGTTGATCGACTAATAAAG ACTTGCATGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTACT AGTCGACGCGTGGCT

31	TeAr154 N	511	TGATCCCTGCACGGCGAATCCACCTGGCAGGTACTCAACGAACTTC AGGACACATTCTTCACCTGGACACGGGAAACTGGCAACAAGCAGAA TGCACTGTCTCCCAGTCTTCGTCATTCTTCTGCTACTGACTGCATC TGGACTTAGCGTTGATGCCCGACCGAAGACCGAAGATGATGTGCCC CTGTCATCTTTCCGTGATAATACAAAGAGTACCCTACAAAGACTTT TGAAGCGAGTCAACTGCTGTCCTATTGATGAATCTTGCTGTTCTTA ACCAGCATGAAGGGAAATGACTTTGGATGAGACCCCTGCGAACTGT TCTGGATGTGAAATTTGGAAAGCAGACTGTTCCTTTCGCACGTGTT CGTGGAATTTCGAATGGTCGTTAACAACACGCTTGCACTTGCAAGC TACTATCTTTCTGTCCTTTCATCTGTGGAACTGTATTGATCAAGTA ACTGAAATGTCATAAAAAAAAAAAAAAAAAGTACTAGTCGACGCGT GGCCA
				32	TeAr193 N	558	CCTGGCAGGTACTCAACGAACTTCAGGACACATTCTTTACACCTGG ACACGGGAAACTGACTACAAGCAGAATGCGCTGCCTCCCAGTCTTC GTCATTCTTCTGCTGTTGATTGCATCTGCGCCTAGCGTTGACGCCC AACCGAAGACCAAAGATGATATACCCCAGGCATCTTTCCTAGATAA TGCGAAGCGATACCTGCAAGTACTTGAGAGCAAAAGAAATTGCTGC AGAAGGCAAATTTGCTGTGGGAGAACAAAATAAATGACGTTGGTTG AGACCCCTGCAAACTGTCCCTGGATGCGAGATTTGGAAAGCAGACT GTTCCTTTCGCACGTGTTCGTGGAATTTCGAATGGTCGTTAACCAC ACGCTGCCACTTGCAAGCTACTATCTCTCTGTCCTTTCATCTGTGG AACTGTATGATCAAACAACTGAAATGTCATATAAATTTTTCAGTGG GTAAACACTATGATCATGTAGTCAGTAATTACATCATTTGGACTTT CTGAAATATTTTTCAGTATGTAAGTGTGTTCCCTTGAAAAGGTCCT TTGTAG

33	LeDr192 N	693	CCTGGCAGGTACTCAACGAACTTCAGGACACATTCTTTTCACCTGG ACACAGGAAGCTGACTACAAGCAGAATGCGCTGTTTCCCAGTCTTC ATCATTCTTCTGCTGCTAATTGCATCTGCACCTTGCTTTGATGCCC GAACGAAGACCGATGATGATGTGCCCCTGTCACCTCTCCGCGATAA TCTAAAGCGAACGATACGAACACGCCTGAACATACGCGAGTGCTGC GAGGATGGATGGTGCTGTACTGCTGCACCCTTAACAGGTCGTTAGG GATAAAGGAAAATGGCTTTGGATGAGACCCCTGCGAATTGTCCCTG GATGTGAGATTTGGAAAGCAGACTGTTCCTTTCGCACGTGTTCGTG GAATTTCGAATGGTCGTTAACAACACGCTGCCACTTGCAAGCCACT ATCTCTCTGTCCTTTCGTATGTGGAACTGTATGATCTAACAACTGA AATGTCAGAAAGTTTTCAGTGGGTATACACTATGATCGTATAGTCA GTAATTACATCATTTGGACTTTCTGAAATATTTTTCGAAATGTGTT TTCCCTTGTAAAGGTCCTTAGTAATTATATTTTAGTGTGTTATGTT TTCCATACAAGTTATAGAATGCTGTCTTTCCTTTTGTTCCCACATC AATGGTGGNGACATAAATTATTGGTTTTGATCAATGTAATTATGAC CTT
				34	LeDr243 N	456	GGAAGCTGACTACAAGCAGAATGCGTTGCCTCCCAGTCTTCGTCAT TCTTCTGCTGCTGATTGCATCTACACCTAGCATTGATGCCCGACCG AAGACCAAAGATGATATGCCCCTGGCATCTTTCAACGATAATGCAA AGCGAATCCTGCAAATACTTTCGAGGAAACCCTGTTGCAGCATTCA CGACAATTCGTGCTGTGGTTTAGGATAAAGGAAAATGACCTTGGAT GAGACCCCTGCAAACTGTCCCTGGATGTGAGATTTGGAAAGCAGAC TGTTCCTTTCGCACGTGTTCGTGGAATTTCGAATGGTCGTTAACAA CACGCTGCCACTTGCAAGCTACTATCTCTCTGTCCTTTCATCTGTG GAACTGTATGATCTAACAACTGAAATGTCGTAGACATTTTTCAATG GGCATACACTATGACCATGTAGTCAGTAATTACATCATTTGA

35	LiC121N	459	GGAAGCTGACTACAAGCAGAATGCGCTGTGTCCCAGTCTTCATCAT TCTTCTGCTGCTGAGTCCATCTGCACCTAGCGTTGATGCCCATCCG AAGACCAAAGATGATGTGCCCCTGGCATCATTCCATGATGATGCAA AGCGAACCCTACAACGACTTTGGATAAAAGCCTTGTGCTGCTACGG ATATAGATTTTGTTGCCCCAATTTTCGGTAAAGGAAAATGATTTTG AATGAGACCCCTGCGAACTGTCCCTGGATGTGAAATTTGGACTGCA GACTGTTCCTTTCGCACGTGTTCGTGGAATTTCGAATCGTCGTTAA CAATACGCTGCCATTTGCAAGCTATTATCTCTCTGTCCTTTCATCT GTGGAACTTAATGATCCAACAACTGAAATGTCACTGAAATTCTTCA AAGGACGACTACTATGATCATGTAGTCAGTAATTACATCATTTGA
				36	MaI126N	460	GGAAGCTGACTACAAGCAGAATGCGCTGTCTCCCAGTCTTGATCAT TCTTCTGCTGCTGACTGCATCTGCACCTGGCGTTGTTGTCCTACCG AAGACCGAAGATGATGTGCCCATGTCATCTGTCTACGGTAATGGAA AGAGTATCCTACGAGGAATTCTGAGGAACGGTGTTTGCTGTGGCTA TAAGTTGTGCCATCCATGTTAACCAGCATGAAGGGAAATGACTTTG GATGAGACCCCTGCGAACTGTCCCTGGATGTGAAATTTGGAAAGCA GACTGTTCCTTTCGCACGTATTCGTGGAATTTCGAATGGTCGTTAA CAACACGCTGCCACTTGCAGGCTACTATCTCTCTGTCCTTTCATCT GTGGAAATGGATGATCTAACAACTGAAATATCATAGAAATTTTTCA ATGGCTATACACTATGACCATGTAGTCAGTAATTACATCATTTGA

Embodiment 2 T-superfamily conotoxin BeB34M (SEQ ID NO:2) and LiC33M's (SEQ ID NO:4) is synthetic

According to the aminoacid sequence of conotoxin peptide BeB34M and LiC33M, this two peptide species that adopted Fmoc method synthetic.Concrete grammar is as follows.

Adopt the Fmoc method in the solid-phase synthesis, on ABI Prism 433a Peptide synthesizer, synthesized above-mentioned two conotoxin peptides.The amino acid whose Side chain protective group of Fmoc is: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr); OBut (Asp); Boc (Lys). adopt Fmoc HOBT DCC method, Rink resin and Fmoc amino acid, the synthetic handbook of synthesis step reference instrument carries out.For reacting completely, distinguish proper extension at the piperidines deprotection and on the coupling time, difficulty is connect amino acid adopt two couplings.Reclaim the linear peptides crude product, use 250*4.6mm, Hypersil ODS-2 column purification, linear peptides purity reaches more than 88%, and identifies by mass spectrum (MS).The molecular weight of BeB34M and LiC33M is respectively 1184.41 and 1658.03.It is folding to be used for oxidation after the linear peptides freeze-drying.

Embodiment 3 conotoxin BeB34M and the oxidation of LiC33M linear peptides are folding

The reductibility linear peptides BeB34M of purifying and LiC33M be the folding 24h of selective oxidation at room temperature.The folding damping fluid of oxidation contains 1mM reduced form (GSH) and 0.5mM oxidized form (GSSG) Triptide (pH 7.5).With the folding peptide of liquid chromatography (LC) system (Bio-Rad) purifying oxidation.Mixture after oxidizing reaction finishes is written into a preparative chromatography post, and (4.6 * 250mm, SinochromODS-BP), flow velocity 1ml/min uses the 0.1%TFA wash-out, and is complete by elution up to all reaction solutions.Solvent orange 2 A is the acetonitrile solution of 0.1%TFA, and solvent B is the aqueous solution of 0.1%TFA.Gradient elution (10% → 100%A, 90% → 0%B, 30 minutes) can be purified into 96% the oxidation bioactive peptide after folding.

The test of embodiment 4 BeB34M anti-insect activity.

With reference to Xiu-hong Wang, Ross Smith et al, Structure-functionstudies of ω-atracotoxin, a potent antagonist of insectVoltage-gated calcium channels.Eur.J.Biochem.264, the method of 488-494 (1999), the anti-insect activity of test b eB34M. BeB34M is expelled to cockroach (Periplaneta americana) with different concentration, oriental tobacco budworm (Heliothis assultaGuenee), in larva of lichee stinkbug (Tessaratoma papillosa) or nymph (body weight 30-180mg) body, observe the response situation of polypide in the 48h.Every insect is injected 5 μ L to belly central authorities with microsyringe, and polypide is temporarily put and prevents insect motion on ice before the injection.5 μ L distilled water (ddH are injected in contrast simultaneously ₂O) or 5 μ L insect physiological saline. two kinds of toxin are used insect physiological saline (200mM NaCl, 3.1mM KCl, 5.4mM CaCl respectively ₂, 5mM MgCl ₂, 2mM NaHCO ₃, 0.1mM NaH ₂PO ₄, pH 7.2) and preparation.Toxin concentration is 10-10 ³Pmol (every gram body weight).Every group of 10 insects, the mortality ratio in the record injection back 48h.Calculate insect medial lethal dose LD50.

Calculation formula is: y=(a-b) LD50/x LD50=xy/ (a-b)

Y is the per cent death loss of injection back 48h sample colony, and x is toxic dose (pmolg ^-1), a is the maximum reactant amount, b is a minimum reacting dose.LD50 is the insect medial lethal dose.

The result shows that BeB34M is slightly variant to the medial lethal dose LD50 of above-mentioned insect, and the LD50 of BeB34M is respectively LD50 cockroach 135 ± 12pmolg ^-1, LD50 oriental tobacco budworm 106 ± 8pmolg ^-1, LD50 lichee stinkbug 128 ± 9pmolg ^-1BeB34M all has lethal effect to the examination insect.

The analgesic activities of embodiment 5 test LiC33M

Utilize the mouse hot-plate test to measure the analgesic activities of LiC33M SEQ ID NO:4 peptide.

Test is (22g ± 3g) with the kunming mice body weight.Mouse is adopted tricorn or intraperitoneal administration.Before 55 ℃ of hot-plate tests 30 minutes, contain the salts solution (0.9% physiological saline) of different concns LiC33M toxin peptide for every injected in mice 5 μ L, with hot plate method measure mouse foot be heated lick metapedes after the analgesia or lift metapedes and later the time be the threshold of pain time, 60s is 100% analgesia.The reaction of mouse is observed in the injection back.If 5 dose concentrations: 0,5,10,20,30ng/ only, 5 mouse of every dosage.The result shows that (table 6) LiC33M dosage is that 10ng/ analgesic activities only is 50 ± 18s; When dosage during more than or equal to 20ng/, analgesic activities is greater than 60s, so LiC33M has analgesic activities.

Table 6 T-CTX LiC33M peptide is to the activity of mouse

Dosage	0 (ng/ only)	5 (ng/ only)	10 (ng/ only)	20 (ng/ only)	30 (ng/ only)
						Threshold of pain time/s	15±5	35±13	50±18	＞60	＞60

The foregoing description is just in order to illustrate rather than limit the present invention.Those skilled in the relevant art know clearly, can do other suitable modification and variation to content as herein described, and can carry out this modification and variation in the scope of the present invention or its any embodiment.Such modification and variation all fall into protection scope of the present invention.

Claims (12)

1. T-superfamily conotoxin peptide, it comprises the aminoacid sequence that is selected from down group:

(1) aminoacid sequence shown in arbitrary in SEQ ID NO:1～12;

(2) aminoacid sequence shown in arbitrary in SEQ ID NO:13～24;

(3) with above-mentioned (1) or (2) described aminoacid sequence at least 80%, preferred about at least 85%, more preferably about at least 90%, especially preferred about at least 95%, most preferably about at least 97% identical aminoacid sequence; Or

(4) because of 1-5, preferred 1-3, more preferably 1-2, most preferably replacement, disappearance, insertion and/or the interpolation and above-mentioned (1) or the different aminoacid sequence of (2) described aminoacid sequence of 1 amino-acid residue.

2. the conotoxin peptide of claim 1, it has and is selected from the aminoacid sequence shown in arbitrary in SEQ ID NO:1～24:

3. the polynucleotide of coding claim 1 or 2 described T-superfamily conotoxin peptides.

4. the polynucleotide of claim 3, it comprises the nucleotide sequence that is selected from down group:

(1) nucleotide sequence of arbitrary described aminoacid sequence in coding SEQ ID NO:1～12;

(2) nucleotide sequence of arbitrary described aminoacid sequence in coding SEQ ID NO:13～24;

(3) nucleotide sequence of SEQ ID NO:25-36 shown in arbitrary;

(4) SEQ ID NO:25-36 arbitrary shown in corresponding mature polypeptide coding sequence in the nucleotide sequence; Or

(5) under rigorous condition can with the nucleotide sequence of above-mentioned (1), (2), (3) or (4) described nucleotide sequence hybridization.

5. a nucleic acid construct wherein comprises claim 3 or 4 described polynucleotide, and one or more control sequences that can be operatively connected, can instruct polypeptide to produce in suitable expressive host with it.

6. an expression vector wherein comprises the described nucleic acid construct of claim 5.

7. a cell transformed has wherein transformed the nucleic acid construct of claim 5 or the expression vector of claim 6.

8. the cell of claim 7, it is a for example bacterial cell of prokaryotic cell prokaryocyte, perhaps eukaryotic cell yeast cell for example, or vegetable cell, for example wheat, corn, paddy rice, soya cells etc.

9. pharmaceutical composition wherein comprises claim 1 or the 2 described conotoxin peptides and the pharmaceutically acceptable carrier of pharmacy effective dose.

10. insecticides wherein comprises claim 1 or the 2 described conotoxin peptides and the optional carrier of insecticidal effective dose.

11. a fusion rotein, other aminoacid sequence that wherein comprises claim 1 or 2 described conotoxin peptides and merge with it.

12. the method for a kill pests comprises the insecticides that the site of containing described insect is used claim 10.