CN1796414A - New alpha - conantokins, coded polynucleotide and application - Google Patents
New alpha - conantokins, coded polynucleotide and application Download PDFInfo
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- CN1796414A CN1796414A CNA200410103563XA CN200410103563A CN1796414A CN 1796414 A CN1796414 A CN 1796414A CN A200410103563X A CNA200410103563X A CN A200410103563XA CN 200410103563 A CN200410103563 A CN 200410103563A CN 1796414 A CN1796414 A CN 1796414A
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Abstract
This invention describes a new alpha-CTX, its encoding polynucleotide, the construction units and transformation cells having this encoding polynucleotide, and the reconstruction process for producing the said alpha-CTX. This invention also describes the artificial synthesis and applications of the said alpha-CTX.
Description
Technical field
The present invention relates to new alpha-conotoxin peptides, the polynucleotide of this peptide of encoding contain the construct and the host cell of these polynucleotide and the method for synthetic and the described peptide of recombinant production.The invention still further relates to the purposes of described conotoxin peptide.
Background technology
Cone shell (Conus) belongs to the preceding cheek subclass Conidae of Gastropoda, is the carnivorous mollusk of a class.500 kinds of cone shells are arranged on the earth approximately, and (Conotoxin, CTX), and the toxin peptide of different cone shell kinds is different for the different conotoxin of the 50-200 kind of having an appointment in every kind of cone shell venom.The specific function that has at least several ten thousand kinds of cone shell phallotoxins to have to regulate various ionic channels, CTX can distinguish the different subtype of various ionic channels, is widely used in neuropharmacological research and as diagnostic reagent and medicine.Thereby the cone shell venom being referred to as " marine drug treasure-house ", they are a kind of unlimited drug resources that are close to.Conotoxin is the focus of current marine drug research and development, enjoys the concern of world's pharmacy circle.Conotoxin chemical structure novelty, biological activity is strong, and the selectivity height of target site has become the new source of the strong instrument and the new drug development of pharmacology and neuroscience.The carnivorism conotoxin is selectively benumbed and lethal effect multiple worm, has broad application prospects at polypeptide sterilant development field.The cone shell that lives in the tropic sea midocean exists only in the South Sea in China, is the distinctive medicinal Living marine resources in Hainan.
Act on biological intravital different target position according to conotoxin and can be divided into 3 classes: (1) acts on the CTX of ligand-gated ion channel, comprises 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.(2) act on the CTX of voltage gated ion channel, voltage gated ion channel claims the voltage sensitivity passage again, often names with penetrating ion (as Na+, K+, Ca2+ etc.).(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.According to the conservative property of 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.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).
Ligand-gated ion channel is the embrane-associated protein that the quick cynapse of mediation is transmitted, many these proteinoids are cloned, can classify according to the similarity of its structure and function, wherein there is a big class to belong to same gene family, be by vagusstoff (acetylcholine), the varies in the blood (serotonin) is as serotonin-3 acceptor (5HT3), γ-An Jidingsuan (GABA), Padil (glycine) activated.Channel protein complex body by function is made up of 5 subunits, and each subunit contains four transbilayer helixs.Except its ligand specificity, on to penetrating ion selectivity, still have any different.The ligand-gated ion channel of other gene families is glutamate receptors, can be subdivided into NMDA (N-methyl-D-aspartate N-methyl-D-aspartate) acceptor and non-NMDA receptor (kainate/AMPA) usually.The 3rd class ligand-gated ion channel is to connect the relevant ATP acceptor of mediator with some neural line.
In CTX, be having of target: act on α-CTX, α A-CTX and the ψ-CTX of n-AChR acceptor, act on the σ-CTX of 5HT3 acceptor and act on the conantokins of nmda receptor with the ligand-gated ion channel.Wherein α-CTX (C C-C-C), α A-CTX (CC-C-C-C-C) belong to the A superfamily, have different halfcystine patterns.ψ-CTX (CC-C-C-CC) belongs to the M superfamily.That wherein study at most is α-CTX.α-CTX comprises GI, GIA, Deng, they are broadly similar structurally: conservative halfcystine skeleton, act on the n-AChR of muscle class, generally contain 13～15 amino acid, be mainly derived from the ichthyophagy cone shell, as ground-tint cone shell (C.geographus), unreal cone shell (C.magus), strain line cone shell (C.striatus) etc.PnIA and PnIB are the CTX of α A-CTX class, derive from food mollusk cone shell seat cone shell (C.pennaceus).And ImI is ψ-CTX of a kind of n-AChR of acting on, derive from the grand cone shell of carnivorism cone shell (C.imperialis), they have identical halfcystine skeleton with the α-CTX in the ichthyophagy cone shell, but the sequence pattern difference, the target of effect is the neurone subclass of n-AChR.
Different α-CTX differs several magnitude sometimes to the affinity difference of vertebrates acceptor.Difference between this all system makes α-CTX can be used as the kind system generation that useful probe is used to study vertebrates n-AChR, can be used as the different subtype that molecular probe is determined nAchR; α-CTX designs new drug as molecular model; As the instrument medicine of research nervous system disease such as Parkinson's disease, take action obstacle, schizophrenia etc., inquire into pathogeny.Alpha-conotoxin can also in conjunction with and the nAchR on blocking-up small cell lung cancer surface, in the diagnosis of small cell lung cancer and treatment, potential using value is arranged.Carnivorism α-CTX can be developed as the polypeptide sterilant.
Fainzilber etc. studied α A-PnIA and α A-PnIB, and its characteristic is different from α-CTX, and there is single negative charge residue in the maximum C end that is not both.CTX with other compares, and it has bigger selectivity to vertebrate skeletal muscle n-AChR.ψ-CTX also suppresses n-AChR, but it does not block the binding site of α-Yin ring toxin (competitive n-AChR antagonist), proves that ψ-CTX does not combine with AChR.Its reason may be the disulfide linkage of this toxin connect with α-CTX or-α A CTX different, and to play inhibiting μ-CTX similar with the Na+ passage.
α-CTX acts on the acetylcholine receptor of teleneuron specifically, has α-CTX in the cone shell poison pipe of most of kinds, estimates to have at least in the venom of Conus α-CTX different in 1000.So far α-the CTX that has found and studied or clinical value is arranged only ten plants one of not enough percentage.The still undiscovered and research of most α-CTX (more than 99%).Therefore the new α-CTX of research and development is significant.
Summary of the invention
What the present invention is based on is the novel alpha-conotoxin with medicinal function found respectively the wart wormwood artemisia cone shell (Conus lividus Hwass) that produces from China Hainan, conus (Conus littertus Linnaeus), the picture-weaving in silk cone shell (C.textile Linnaeus) (α-Conotoxin, the peptide of α-CTX).
Therefore, in one aspect, the invention provides the new alpha-conotoxin (peptide of α-CTX).
In yet another aspect, the invention provides the polynucleotide of this peptide of coding.
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, bollworm, oriental tobacco budworm, the snout moth's larva of rice, sugarcane moth borers, and they are expelled in the mouse body, the no abnormality seen reaction.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 have analgesic activities by playing a role in conjunction with acetylcholine receptor (nAChR).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.
Embodiment
In one embodiment, the invention provides alpha-conotoxin peptides, it comprises the aminoacid sequence that is selected from down group:
(1) aminoacid sequence shown in arbitrary among the SEQ ID NO:1-3;
(2) aminoacid sequence shown in arbitrary among the SEQ ID NO:4-6;
(3) aminoacid sequence identical with aminoacid sequence shown in above-mentioned (1) or (2) 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 the different aminoacid sequence of sequence shown in above-mentioned (1) or (2) of 1 amino-acid residue.
Preferably, the aminoacid sequence that described conotoxin peptide has is identical with arbitrary aminoacid sequence about at least 85% among the SEQ ID NO:1-6, more preferably identical greater than 90% at least, 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-p blastp-a4-e10-E0-v500-b250-I[inquires about document]-d prot_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.
Arbitrary aminoacid sequence difference 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 among the aminoacid sequence of homeopeptide and the SEQ ID NO:1-6.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:1-6 arbitrary shown in the nucleotide sequence of polypeptide of aminoacid sequence.In a further preferred embodiment, these polynucleotide comprise among the SEQ ID NO:7-9 nucleotide sequence shown in arbitrary or corresponding mature polypeptide coding sequence wherein.The present invention also comprise coding have among the SEQ ID NO:1-6 arbitrary shown in the nucleotide sequence of polypeptide of aminoacid sequence, different between the corresponding mature polypeptide coding sequence among it and the SEQ ID NO:7-9 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:7-9 in the mature polypeptide coding sequence of arbitrary sequence 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:7-9, 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:7-9 in the mature polypeptide coding sequence of arbitrary sequence 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, 5XDenhardtShi 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 takes place as the kind system that useful probe is used to zoologize nAChR; Determine the different subtype of nAchR as molecular probe; As molecular model, the design new drug; Instrument medicine as research nervous system disease such as Parkinson's disease, take action obstacle, schizophrenia etc.; The candidate medicine of treatment small cell lung cancer; As the polypeptide sterilant, be developed as novel biopesticide etc.
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 conotoxin gene
The extraction of the total RNA of 1 cone shell
With wart wormwood artemisia cone shell (Conus lividusHwass), conus (Conus littertus Linnaeus) and picture-weaving in silk cone shell (C.textileLinnaeus) live body from coastal collections such as Hainan Island, the Xisha Islands is 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.
2cDNA's is synthetic
Adopt AMV Transcriptase test kit (Invitrtogen), the total RNA that extracts with step 1 is a template, and Oligo (dT) 15 is a primer, synthetic cDNA.
The reacted constituent of synthetic cDNA is: 0.5ug Oligo (dT) 15, the total RNA of 2ug, 1 * reverse transcription damping fluid, 1mM dNTP, 20U recombinant RNA enzyme inhibitors ribonuclease inhibitor (Recombinant Rnasin Ribonuclease Inbibitor), 25U AMV ThermoScript II, redistilled water.The reaction cumulative volume is 20uL.Response procedures: earlier with total RNA 70 ℃ of incubations 10 minutes, add other reacted constituents again; In 42 ℃ of incubations 1 hour, 95 ℃ of termination reactions 5 minutes; Reverse transcription product is put-20 ℃ of preservations.Get the 5ul reverse transcription product and carry out the detection of 1% agarose electrophoresis.
3 reverse transcription PCRs reactions (RT-PCR)
With step 2 synthetic cDNA is template, according to A-superfamily conotoxin signal peptide zone conserved regions and 3 ' non-translational region sequence (J.Michael McIntosh, Cheryl Dowell et al. α-Conotoxin GIC from Conus geographus, a Novel Peptide Antagonist of NicotinicAcetylcholine Receptors.J.Biol.Chem., 2002,277, (37): 33610-33615.Michael Ellison, J.Michael McIntosh, and Baldomero M.Olivera. α-ConotoxinsImI and ImII SIMILAR alpha7 NICOTINIC RECEPTOR ANTAGONISTS ACT AT DIFFERENTSITES.J.Biol.Chem., 2003,278 (2): 757-764.), design α-CTX primer (upstream primer 5 ' TCT G ATG GCA GGA ATG ACG CAG 3 ' (SEQ ID NO:10), downstream primer 5 ' TCG TGG TTC AGA GGG TCC TGG 3 ' (SEQ ID NO:11)), carry out the PCR reaction.PCR reaction cumulative volume is 25ul, comprising: cDNA template 50ng, 10 * PCR buffer, 200uM dNTPs, 1umol upstream and downstream primer, 2mM MgCl2,1UTaq enzyme and deionized water.Cycling program: 94 ℃ of pre-sex change 3 minutes, 94 ℃ of sex change 30 seconds, 50 ℃ (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 4PCR product and order-checking
Reclaim above-mentioned specific PCR product, be connected back transformed into escherichia coli XL1 bacterial strain 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, three kinds of novel α of the present invention-CTX precursor-gene LiC22N (SEQ ID NO:7), LeD2N (SEQ IDNO:8) and TeA21N (SEQ ID NO:9) have been obtained.
According to precursor-gene and conotoxin characteristics, infer conotoxin propetide LiC22P, LeD2P and TeA21P that they have the aminoacid sequence shown in the SEQ ID NO:4,5 and 6 respectively; Infer according to propeptide sequence mature peptide LC22M, LeD2M and TeA21M, they have the aminoacid sequence shown in the SEQ ID NO:1,2 and 3 respectively again.The aminoacid sequence of the nucleotide sequence of three kinds of genes, coded precursor peptide and corresponding mature peptide is as follows: LC22N (α-CTX gene, 148bp is from wart wormwood artemisia cone shell (Conus lividus Hwass, Livid Cone))
TCTGATGGCAGGAATGACGCAGCCAACGACAAAGCGTCTAA
ACTGATGGTTCTTAGGAACGAATGCTGTGACAATCCTCCGTG
CAAGTCGAGTAATCCAGATTTGTGTGACTGGAGAAGCTGATG
CTCCAGGACCCTCTGAACCACGA(SEQ ID NO：7)。
Propetide LiC22P by the LiC22N coding is made up of 40 amino acid, and sequence is: SDGRNDAANDKASKLMVLRNECCDNPPCKSSNPDLCDWRS (SEQ ID NO:4).
Be made up of 21 amino acid through the mature peptide LiC22M that processing produces propetide LiC22P, sequence is NECCDNPPCKSSNPDLCDWRS (SEQ ID NO:1).LeD2N (α-CTX gene, 151bp is from conus (Conus litteratusLinnaeus, Lettered Cone))
TCTGATGGCAGGAATGACGCAGCCAGCAACAAAGCGTCTCA
CCTGATCGCCCTGGCCGTCAGGGGATGCTGTGCCCGTGCTG
CCTGTGCCGGGATTCATCAAGAACTTTGTGGAGGAGGACGC
TGATGCTCCAGGACCCTCTGAACCACGA(SEQ ID NO：8)。
Propetide (LeD2P) by the LeD2N coding is made up of 41 amino acid, and sequence is: SDGRNDAASNKASHLIALAVRGCCARAACAGIHQELCGGGR (SEQ ID NO:5).
Be made up of 16 amino acid through the mature peptide LeD2M that processing produces propetide LeD2P, sequence is:
GCCARAACAGIHQELC# (SEQ ID NO:2, # represent this toxin carboxyl terminal by amidation).
TeA21N (α-CTX gene, 151bp is from picture-weaving in silk cone shell (C.textile Linnaeus, Textile Cone))
TCTGATGGCAGGAATGACGCAGCCAAAGCGTCTGGCCTGGT
CAGTCTGACTGACAGGAGACCAGAATGCTGTAGTGATCCTCG
CTGTAACTCGAGTCATCCAGAACTTTGTGGTTGACGACGCTG
ATGCTCCAGGACCCTCTGAACCACGA(SEQ ID NO：9)。
Propetide (TeA21P) by the TeA21N coding is made up of 38 amino acid, and sequence is: SDGRNDAAKASGLVSLTDRRPECCSDPRCNSSHPELCG (SEQID NO:6).
Be made up of 17 amino acid through the mature peptide TeA21M that processing produces propetide TeA21P, sequence is:
PECCSDPRCNSSHPELC# (SEQ ID NO:3, # represent this toxin carboxyl terminal by amidation).
Embodiment 2 conotoxin LC22M, LeD2M's is synthetic
According to the aminoacid sequence of conotoxin mature peptide LC22M, LeD2M, 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 two conotoxin peptides of LC22M, LeD2M.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 85%, and identifies by mass spectrum (MS).The molecular weight of LC22M is 2382.59; The molecular weight of LeD2M is 1604.90.Be used to after the freeze-drying fold.
Embodiment 3 conotoxin LC22M, the oxidation of LeD2M linear peptides fold
The abundant reductive LC22M of 10 μ M, LeD2M linear peptides are at folding damping fluid (100mM NH
4HCO
3(pH 8.0), 23-25 ℃) in stir folding 24-48h. and use liquid chromatography (LC) (Bio-Rad) system purifying again through the folding peptide of oxidation.
The test of embodiment 4LiC22M anti-insect activity.
With reference to Xiu-hong Wang, Ross Smith et al, Structure-functionstudies of ω-atracotoxin, a potent antagonist of insect voltage-gatedcalcium channels.Eur.J.Biochem.264, the method of 488-494 (1999), the anti-insect activity of test LiC22M. LiC22M is expelled to cockroach (Periplaneta americana) with different concentration, bollworm (Heliothis armigera Huber), oriental tobacco budworm (Heliothis assulta Guenee), the snout moth's larva of rice (yellow rice borer, Tryporyza incertulas (Walker),), in larva (body weight 50-180mg) body of sugarcane moth borer (Chilo infuscatellus Snellen), 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 .LiC22M toxin with insect physiological saline (200mM NaCl, 3.1mM KCl, 5.4mM CaCl
2, 5mM MgCl
2, 2mM NaHCO
3, 0.1mM NaH
2PO
4, pH 7.2) and preparation.Toxin concentration is 10-103pmol (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 LiC22M is more similar to the medial lethal dose LD50 of above-mentioned insect, is respectively LD50 cockroach 95 ± 10pmolg
-1, LD50 bollworm 86 ± 8pmolg
-1, LD50 oriental tobacco budworm 76 ± 6pmolg
-1, the LD50 snout moth's larva of rice 92 ± 9pmolg
-1, LD50 sugarcane moth borer 82 ± 7pmolg
-1When LiC22 concentration reaches 200pmolg
-1The time, the insect mortality that tries is 100%, and the control group mortality ratio causes because of injection operation more than indivedual contrasts are dead less than 5%.Therefore LiC22M all has very strong lethal effect to the examination insect.
| The insect type | Cockroach | Bollworm | Oriental tobacco budworm | The snout moth's larva of rice | Sugarcane moth borer |
| LD50 value (pmolg -1) | 95±10 | 86±8 | 76±6 | 92±9 | 82±7 |
The analgesic activities of embodiment 5 test LeD2M
Utilize the mouse hot-plate test to measure the analgesic activities of LeD2M.
Test is (20g ± 3g) with the kunming mice body weight.Mouse is adopted the tricorn administration, and every injection 20 μ L contain the LeD2M salts solution (150mM NaCl) of different toxin concentrations, 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.If 4 dose concentrations: 0,5,10,20ng/ only, 5 mouse of every dosage.The result greater than 10ng/ only shows LeD2M dosage, and analgesic activities (hot plate method) is greater than 60s, and more than the effect 4h.Show that analgesic activities is powerful.
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. conotoxin peptide, it comprises the aminoacid sequence that is selected from down group:
(1) aminoacid sequence shown in arbitrary among the SEQ ID NO:1-3;
(2) aminoacid sequence shown in arbitrary among the SEQ ID NO:4-6;
(3) with aminoacid sequence at least 80% shown in above-mentioned (1) or (2), preferred at least 85%, more preferably at least 90%, especially preferred at least 95%, most preferably 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 the different aminoacid sequence of sequence shown in above-mentioned (1) or (2) of 1 amino-acid residue.
2. the conotoxin peptide of claim 1, it has the aminoacid sequence of the group of being selected from down:
(1) aminoacid sequence shown in arbitrary among the SEQ ID NO:1-3; Or
(2) aminoacid sequence shown in arbitrary among the SEQ ID NO:4-6.
3. the polynucleotide of coding claim 1 or 2 described conotoxin peptides.
4. the polynucleotide of claim 3, it comprises the nucleotide sequence that is selected from down group:
(1) coding SEQ ID NO:1-3 arbitrary shown in the nucleotide sequence of aminoacid sequence;
(2) nucleotide sequence of the arbitrary described aminoacid sequence of coding SEQ ID NO:4-6;
(3) nucleotide sequence shown in arbitrary among the SEQ ID NO:7-9;
(4) arbitrary corresponding mature polypeptide coding sequence that comprises among the SEQ ID NO:7-9; 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, or eukaryotic cell yeast cell for example, or vegetable cell, for example wheat, corn, paddy rice or soya cells.
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 the conotoxin peptide of claim 1 or 2 and merge mutually with it.
12. the method for a kill pests comprises the insecticides that the site of containing described insect is used claim 10.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381403B (en) * | 2008-09-17 | 2011-03-16 | 中国人民解放军军事医学科学院生物工程研究所 | Alpha type conotoxin peptide derivates and use thereof |
| CN102286079A (en) * | 2010-03-10 | 2011-12-21 | 中国人民解放军军事医学科学院生物工程研究所 | Alpha type conotoxin peptides and applications thereof |
| CN102875653A (en) * | 2011-07-15 | 2013-01-16 | 海南大学 | Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof |
| CN102876683A (en) * | 2012-10-24 | 2013-01-16 | 中国农业科学院生物技术研究所 | Conotoxin and biological preparation method and application thereof |
| CN103374066A (en) * | 2012-04-19 | 2013-10-30 | 海南大学 | Alpha B-O-conotoxin and medicine composition and application thereof |
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|---|---|---|---|---|
| CN1169830C (en) * | 1999-04-30 | 2004-10-06 | 解放军军事医学科学院生物工程研究所 | Conotoxin peptide |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381403B (en) * | 2008-09-17 | 2011-03-16 | 中国人民解放军军事医学科学院生物工程研究所 | Alpha type conotoxin peptide derivates and use thereof |
| CN102286079A (en) * | 2010-03-10 | 2011-12-21 | 中国人民解放军军事医学科学院生物工程研究所 | Alpha type conotoxin peptides and applications thereof |
| CN102286079B (en) * | 2010-03-10 | 2013-06-19 | 中国人民解放军军事医学科学院生物工程研究所 | Alpha type conotoxin peptides and applications thereof |
| CN102875653A (en) * | 2011-07-15 | 2013-01-16 | 海南大学 | Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof |
| CN102875653B (en) * | 2011-07-15 | 2015-04-01 | 海南大学 | Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof |
| CN103374066A (en) * | 2012-04-19 | 2013-10-30 | 海南大学 | Alpha B-O-conotoxin and medicine composition and application thereof |
| CN103374066B (en) * | 2012-04-19 | 2015-02-11 | 海南大学 | Alpha B-O-conotoxin and medicine composition and application thereof |
| CN102876683A (en) * | 2012-10-24 | 2013-01-16 | 中国农业科学院生物技术研究所 | Conotoxin and biological preparation method and application thereof |
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