CN103374066A

CN103374066A - Alpha B-O-conotoxin and medicine composition and application thereof

Info

Publication number: CN103374066A
Application number: CN2012101179289A
Authority: CN
Inventors: 罗素兰; 长孙东亭; 吴勇; 胡远艳; 朱晓鹏; J·迈克尔·麦金托什
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2012-04-19
Filing date: 2012-04-19
Publication date: 2013-10-30
Anticipated expiration: 2032-04-19
Also published as: CN103374066B

Abstract

The invention belongs to the field of biological chemistry and molecular biology, and relates to novel alpha B-O-conotoxin, a medicine composition of the novel alpha B-O-conotoxin as well as a preparation method and an application of the novel alpha B-O-conotoxin. The invention also relates to propeptide of conotoxin, a nucleic acid construct of the propeptide, an expression vector and transformed cells of the propeptide, and a fusion protein of the propeptide. The invention also relates to a method for blocking an acetylcholine receptor, and an application of the conotoxin in medicine preparation. The novel alpha B-O-conotoxin can specifically block the acetylcholine receptor (nAChRs) (such as alpha 9 alpha 10nAChR), shows the activity on treating neuralgia as well as the activities on cancer chemotherapy, breast cancer, lung cancer, wound healing and the like and has a good application prospect in preparation of analgesia medicaments, cancer therapy-related medicaments, tool medicaments in neurosciences and other medicaments.

Description

α B-superfamily conotoxin peptide, its pharmaceutical composition and purposes

Technical field

The invention belongs to biological chemistry and biology field, relate to a kind of new α B-superfamily conotoxin peptide, its pharmaceutical composition, Preparation Method And The Use.The invention still further relates to propetide, its nucleic acid construct, its expression vector and the conversion of described conotoxin peptide cell, with and fusion rotein.The invention still further relates to a kind of method of blockage of acetylcholine receptor and the pharmaceutical applications of described conotoxin peptide.

Background technology

NAChR (nAChRs) is the ubiquitous epicyte protein with important physiological action and clinical study meaning of animal kingdom, the physiological function that it mediates numerous maincenters and peripheral nervous system comprises study, remembers, replys, analgesia and motion control etc.NAChRs activates the release of the various neurotransmitters such as Dopamine HCL, norepinephrine, serotonin, γ-aminobutyric acid.Confirmed that nAChRs is the crucial target spot of Screening Diagnosis and treatment one large class important diseases medicine, these diseases comprise the difficult and complicated cases such as habituation, pain, cancer, amentia, parkinsonism, psychosis, depression, myasthenia gravis.So far the medicine that does not also have symptomatic treatment for above-mentioned disease.Nonselective nAChR agonist such as nicotine commonly used, although can alleviate the symptom of above-mentioned sacred disease, they produce strong side effect to heart and gi tract, and habituation is arranged.Therefore, exploitation is key point (the Livett BG of the above-mentioned disease for the treatment of for the part medicine that the various hypotypes of nAChRs have highly selective, Sandall DW, Keays D, Down J, Gayler KR, Satkunanathan N, Khalil Z.Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor.Toxicon.2006,48 (7): 810-829).Yet the prerequisite that will develop such medicine is, obtain can the various hypotypes of specific combination nAChRs alternative cpd, study and identify meticulous composition and the physiological function of various hypotypes as the instrument medicine, or directly as the medicine of relative disease.In addition, in mammary cancer and small cell lung cancer, the activation of nAChR promotes tumor cell proliferation on the tumor cell membrane, blocks the activation of these acceptors with medicine, can effectively carry out early diagnosis, or treat these calamitous cancers.

NAChRs is assembled into a variety of hypotypes by different α and β subunit, and every kind of hypotype has distinct pharmacological characteristic.Because lack the highly selective ligand compound for various hypotypes, fine structure and the function that study and illustrate various nAChRs hypotypes face lot of challenges.

Studies show that α 9 α 10nAChR are novel targets (McIntosh, J.M. for the treatment of neurodynia medicine; Absalom, N.; Chebib, M.; Elgoyhen, A.B.; Vincler, M., Alpha9 nicotinic acetylcholine receptors and the treatment of pain.Biochemical pharmacology 2009,78 (7), 693-702.Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vcl.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones.Brain research 2005,1059 (2), 149-58.).α 9 α 10nAChR blockers have treatment neurodynia and accelerate the function of injured nerve recovery, may be by immunologic mechanism play a role (Holtman, J.R.; Dwoskin, L.P.; Dowell, C.; Wala, E.P.; Zhang, Z.; Crooks, P.A.; McIntosh, J.M., The novel small molecule alpha9alpha10nicotinic acetylcholine receptor antagonist ZZ-204Gis analgesic.European journal of pharmacology2011,670 (2-3), 500-8.Zheng, G.; Zhang, Z.; Dowell, C.; Wala, E.; Dwoskin, L.P.; Holtman, J.R.; McIntosh, J.M.; Crooks, P.A., Discovery of non-peptide, small molecule antagonists of alpha9alpha10nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonicinflammatory pain.Bioorganic ﹠amp; Medicinal chemistry letters 2011,21 (8), 2476-9.) the 9 α 10nAChR of the α on the keratinocyte play a part very important (Chernyavsky, A.I. in the pathophysiological processes of wound healing; Arredondo, J.; Vetter, D.E.; Grando, S.A., Central role of alpha9acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization.Experimental cell research2007,313 (16), 3542-55).Recently studies show that α 9nAChR subunit is crossed and expressed in breast cancer tissue.α 9 subunit variants affect segmental bronchus transformation and propagation, this subunit have very important significance in the treatment of lung cancer (Chi kova, A.; Grando, S.A., Naturally occurring variants of human Alpha 9nicotinic receptor differentially affect bronchial cell proliferation and transformation.PloS one 2011,6 (11), e27978.).

Craving for tobacco is to be caused by the nicotine in the tobacco (Nicotine), its body inner recipient is exactly nAChR (nAChRs) (Azam L, McIntosh JM.Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.Acta Pharmacol Sin.2009; 30 (6): 771-783.).As everyone knows, smoking almost damages the whole vitals of human body (for example respiratory system, the recycle system, neural system, urinary system and other important organ), and to the special harm of pregnant woman and fetus.According to investigations, smoking is the first killer of American's health, annual because of the dead number of smoking up to people more than 430,000, shelter has first of the cause of death, secondly is that the death toll that alcoholism causes is annual about 100,000 people.Smoking is general especially in China, and harm is more serious, and it is dead because of smoking that 2000 Chinese are arranged every day, and to the year two thousand fifty, this death toll will rise to people's every days 8000.The cause of the death that smoking causes is mainly from lung cancer and tuberculosis, and the medicine that can treat lung cancer will have broad application prospects.

In addition, according to investigations, the crowd of ache influence 1/6 comprises sacroiliitis, neurodynia, swells and ache.Wherein neurodynia affects the crowd of 4-8%.The neuralgic method of existing treatment mainly is the toponarcosis medication, blocks because the pain signal of the generations such as peripheral nerve, neuroplexus, Dorsal root nerve, sympathetic nervous system.But these treatments can only have analgesic effect the short period of time, but can not effect a radical cure neurodynia.A lot of diseases all can cause neurodynia, comprise cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation are hindered, acquired immune deficiency syndrome (AIDS), the head nerves paralysis, drug intoxication, industrial pollution is poisoned, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic congenital sensory neuropathy, acute violent idiopathic neuralgia, extruding neurodynia, vasculitis (vasculitis)/local asphyxia, uremia, children's bile hepatic diseases, the chronic respiratory obstacle, the composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, the Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

Neuralgia medicine take α 9 α 10nAChR as target spot can be brought into play analgesic effect (Vincler by intramuscular injection, M.Wittenauer, S.Parker, R.Ellison, M.Olivera, B.M.McIntosh, J.M.Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10nicotinic acetylcholine receptors.Proc Natl Acad Sci USA, 2006,103 (47): 17880-4.), easier than present business-like ω-CTX MVIIA analgesic-Qi Kaonuo peptide route of administration, the Qi Kaonuo peptide need be built in by program pump and be administered directly to spinal cord in the human body, route of administration is pretty troublesome, this administration pump is very expensive, only limit at present developed country's application such as USA and Europe, be difficult to use (Kress HG in vast developing country, Simpson KH, Marchettini P, Ver Donck A, Varrassi G.Intrathecal therapy:what has changed with the introduction of ziconotide.Pain Pract.2009; 9 (5): 338-47.Burton AW, Deer TR, Wallace MS, Rauck RL, Grigsby E.Considerations and methodology for trialing ziconotide.Pain Physician.2010; 13 (1): 23-33.Wallace MS, Rauck RL, Deer T.Ziconotide combination intrathecal therapy:rationale and evidence.Clin JPain.2010; 26 (7): 635-44).Therefore, but analgesic activity more powerful, and the s-generation CTX new drug of intramuscular injection, will have more wide market.

At present, live in the toxin (conotoxin, conopeptide, conotoxin, CTX) that produces in the carnivorous mollusk cone shell venom in the Tropical Ocean and receive much concern, be used to the systematically specific blockage agent of the various hypotypes of research and development nAChRs.

Conotoxin (conopeptide, conotoxin, CTX) is formed, is rich in the neuropeptide toxin of halfcystine (Cys) mostly by 7-50 amino-acid residue.Conotoxin (peptide) is by the similarity of the endoplasmic reticulum signal peptide sequence of its precursor protein, and halfcystine pattern, be divided into different gene families, so far, all known conotoxins can be divided into 17 superfamilies, be respectively A, C, D, S, M, I1, I2, I3, J, L, O1, O2, O3, P, T, V, Y (Kaas Q, Yu R, Jin AH, Dutertre S and Craik DJ.ConoServer:updated content, knowledge, and discovery tools in the conopeptide database.Nucleic Acids Research (2012) [Ahead of print]).Conotoxin (peptide) can be divided into the multiple pharmacology such as α, ω, μ, δ family by its acceptor target position.Each superfamily can be divided into again α, α A, κ A (A-superfamily) according to acceptor target type, ω, δ, κ, μ O (O-superfamily), and familiesies (hypotype) such as μ, φ, KM (M-superfamily) are (Fig. 1).

Conotoxin synthesizes first larger non-activity polypeptide precursor in vivo, they are comprised of 50-80 amino-acid residue, generally be by signal peptide, 3 parts of N-end or C-CICP and mature peptide form, these signal peptide sequences have conservative property in all superfamily members, the proteolysis signal that between propetide and mature peptide, often contains standard, such as basic aminoacids Methionin (K) or arginine (R) etc., the mature peptide district is except halfcystine, its sequence has highly variable, thereby causes separately action target spot and pharmacological activity difference far away.The propetide district is conducive to secretion (Hidaka, the Y. that the mature peptide oxidative folding forms correct disulfide linkage or is conducive to the hydrophobicity conotoxin; Ohno, M.; Hemmasi, B.; Hill, O.; Forssmann, W.G.; Shimonishi, Y., In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein.Biochemistry 1998,37 (23), 8498-507.; Conticello, S.G.; Kowalsman, N.D.; Jacobsen, C.; Yudkovsky, G.; Sato, K.; Elazar, Z.; Petersen, C.M.; Aronheim, A.; Fainzilber, M., The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors.The Journal of biological chemistry 2003,278 (29), 26311-4.).

As seen, the nAChR particularly novel blocker of α 9 α 10nAChR has important using value for the research of this receptor structure and function, and might be developed as the original medicine with the closely related disease of this receptor.Therefore, need the nAChRs blocker of finding new high specific badly.

Summary of the invention

The inventor has found the new superfamily conotoxin peptide of a class α B-through deep research and creatively work.The new superfamily conotoxin peptide of α B-of the present invention has unique 4 halfcystine patterns " C-CC-C ", and does not contain the propetide district, and this halfcystine pattern and precursor peptide compositing area from all conotoxins of finding in the past is all different.The inventor is surprised to find, it is blockage of acetylcholine receptor specifically, particularly the blocking-up activity to neurodynia drug target, mammary cancer, lung cancer target spot α 9 α 10nAChR is the strongest, has the applications well prospect at aspects such as preparation analgesic, relevant cancer treatment drugs and neuroscience instrument medicines.Following invention is provided thus:

One aspect of the present invention relates to a peptide species, its for or comprise and be selected from each described aminoacid sequence in following (1) to (4):

(1) aminoacid sequence (mature peptide) shown in the SEQ ID NO:1 (hereinafter being also referred to as α B-conotoxin VxXXIIIA or α B-VxXXIIIA or VxXXIIIA);

(2) aminoacid sequence (precursor peptide) shown in the SEQ ID NO:2 (hereinafter being also referred to as α B-conotoxin VxXXIIIA precursor or α B-VxXXIIIA precursor or VxXXIIIA precursor);

(3) with above-mentioned (1) or (2) described aminoacid sequence at least 80%, preferred at least 85%, more preferably at least 90%, especially preferred at least 95%, most preferably at least 97% identical aminoacid sequence (being called in this article " homeopeptide "); Or

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

The below is the precursor peptide open reading frame of α B-superfamily conotoxin VxXXIIIA, and precursor peptide (SEQ ID NO:2) and the mature peptide (SEQ ID NO:1) of coding generation.Arrow refers to the site (G) that the proteolysis of inferring is processed as mature peptide.The precursor peptide of VxXXIIIA only contains signal peptide and 2 zones of mature peptide, and the signal peptide zone indicates with italic, and mature peptide zone (SEQ ID NO:1) indicates with underscore, and terminator codon indicates with " * ".All to form structure by signal peptide, N-end propetide and mature peptide or 3 zones of C-end propetide all different from all conotoxins of finding in the past for these, it is a new superfamily conotoxin, the new superfamily of called after B-, again because this toxin is blocked nAChR, so called after α B-superfamily conotoxin (α B-conotoxin):

ATGGAGACTCTCACTCTACTATGGCGAGCGTCGTCGTCTTGCCTGCTCGTCGTT

↓V R C L E K S GCTCAGTCACTCGCTTCTTCGTCTTCTCGGG GTTAGGTGTTTGGAGAAGAGCGGC

A Q P N K L F R P P C C Q K G P S FGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTGTCAAAAGGGCCCTTCTTTC

A R H S R C V Y Y T Q S R E ＊GCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAGTAA

For one object 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 for inquiry.

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:1-2.Preferably, amino acid change is that character changes less variation, namely be can 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, such as the methionine residues of aminoterminal interpolation; The little connection peptides that reaches about 20-25 residue is arranged; Little extension such as poly Histidine fragment, epitope or the land that maybe can help by changing net charge or other function 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 α-amino-isovaleric acid), die aromatischen Aminosaeuren (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).The aminoacid replacement that usually can not change specific activity 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 α B-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.

Each described polypeptide according to the present invention, it has the aminoacid sequence shown in SEQ ID NO:1 (α B-VxXXIIIA) or the SEQ ID NO:2 (this polypeptide is actually the propetide of α B-VxXXIIIA).

Each described polypeptide according to the present invention, wherein, first halfcystine of the N-terminal among the SEQ ID NO:1 and second halfcystine form disulfide linkage, and the 3rd halfcystine and the 4th halfcystine formation disulfide linkage; Perhaps first halfcystine and the 3rd halfcystine form disulfide linkage, and second halfcystine and the 4th halfcystine formation disulfide linkage; Perhaps first halfcystine and the 4th halfcystine form disulfide linkage, and second halfcystine and the 3rd halfcystine formation disulfide linkage.

Aforementioned polypeptides of the present invention is conotoxin peptide; Particularly, be α B-conotoxin peptide.

Above-mentioned conotoxin peptide can extract from the calamus cone shell (Conus C.Vexillum) that produce in China Hainan.Also can chemosynthesis amino acid sequence (for example method in the reference example 3); Perhaps express its Nucleotide (the preparation reference example 1-2 of nucleotide sequence or direct according to the nucleotide sequence synthetic among the embodiment 2) by the means of gene recombination, obtain polypeptide.Also can be with reference to following method:

Another aspect of the present invention relates to the preparation method of each described polypeptide of the present invention, comprises the steps:

1) on ABI Prism 433a Peptide synthesizer or manual method synthesizing linear polypeptide, the amino acid whose Side chain protective group of Fmoc is: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); Halfcystine fixes a point to form respectively disulfide linkage with Trt or Acm blocking group between corresponding halfcystine;

2) with step 1) in the linear polypeptide that obtains cut down from resin, and with ice ether sedimentation and washing and recycling linear polypeptide crude product, with preparation type reverse hplc C18 post (Vydac) purifying;

3) with step 2) in the product that obtains to carry out two-step oxidation folding.

Of the present inventionly relate in one aspect to a kind of polynucleotide, its code book is invented the aminoacid sequence of each described polypeptide again.

Each described polynucleotide according to the present invention, its for or comprise and be selected from each described nucleotide sequence in following (1) to (4):

(1) nucleotide sequence shown in the SEQ ID NO:3 (full-length cDNA of coding VxXXIIIA precursor protein);

(2) nucleotide sequence shown in the SEQ ID NO:4 (open reading frame (ORF) of coding VxXXIIIA precursor protein);

(3) nucleotide sequence shown in the SEQ ID NO:5 (coding VxXXIIIA mature peptide);

(4) complementary sequence of the complementary sequence of the complementary sequence of SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5; Or

(5) under rigorous condition can with the nucleotide sequence of each described nucleotide sequence hybridization in above-mentioned (1) to (4).

The below is precursor peptide full length cDNA sequence (SEQ ID NO:3) and the open reading frame thereof of α B-superfamily conotoxin VxXXIIIA, and open reading frame (SEQ ID NO:4) represents with underscore:

GACTCCTATTGTCATA

GTTAGGTGTTTGGAGAAGAGCGGCGCGCAACCT AATAAGTTATTTCGTCCGCCGTGCTGTCAAAAGGGCCCTTCTT TCGCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGA GtaaTCCGGGGGGTCCGAAATGTGAAATAAGAAGGCCGAGCATACAACCCTCCTTAACACTCGCCTATTGTAAAAGGGTGTCCTTTGTCAATAGTGGTACTAATGTTGTTTAAAAAATCCGCTGTGATATCTTAGGTGTTCAAGTAGTAGGTTGCACCCCCCTCGAGAGGGTATACCAGCTGGTCGTCCGCCGGCTATTAAGTGATCACTCACCCTTTGTTGATGGTGGTACAAGTTAAAGGATCCCCCGGGCGCGTGGTTTTCTTACCACATTTTTAGAAGTCCCCCCGGCGCCGCCGGTTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

About the hybridization between the polynucleotide, there is in the prior art numerous documents can be for reference, comprise such as 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.More rigorous condition, the complementary degree that forms the duplex requirement is higher.Can pass through the rigorous degree of temperature, concentration and probe concentration, probe length, ionic strength, time etc. 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 usually following carrying out: in Tm-20 ℃ in 0.2X SSPE, 0.1%SDS one time 15 minutes (the rigorous condition of moderate is cleaned).

One or more primers pair of relating in one aspect to again of the present invention, comprise the full-length cDNA polynucleotide sequence SEQ ID NO:3 of VxXXIIIA precursor protein according to it, or the designed primer of open reading frame (ORF) the polynucleotide sequence SEQ ID NO:4 of coding VxXXIIIA precursor protein pair.Described primer obtains the polynucleotide sequence shown in SEQ ID NO:3 or the SEQ ID NO:4 to can be used in pcr amplification.

Of the present inventionly relate in one aspect to a kind of nucleic acid construct, it comprises each described polynucleotide of the present invention again.

Of the present inventionly relate in one aspect to a kind of expression vector, it comprises each described nucleic acid construct of the present invention again.

The cell that relates in one aspect to again a kind of conversion of the present invention, it comprises each described expression vector of the present invention.

Of the present inventionly relate in one aspect to a kind of fusion rotein, it comprises each described polypeptide of the present invention again.

Of the present inventionly relate in one aspect to a kind of pharmaceutical composition, it comprises each described polypeptide of the present invention, perhaps comprises fusion rotein of the present invention again; Alternatively, it also comprises pharmaceutically acceptable carrier or auxiliary material.

The method that relates in one aspect to again a kind of blockage of acetylcholine receptor of the present invention comprises the step of each the described polypeptide of the present invention that uses significant quantity; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

Of the present inventionly relate in one aspect to a kind of method of screening acetylcholine receptor inhibitor or definite acetylcholine receptor subtypes, the method comprises: by the step that in the situation that has and do not exist the candidate compound existence each described polypeptide of acetylcholine receptor and the present invention is contacted again; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.When α B-conotoxin VxXXIIIA can specific blockage α 9 α 10 acetylcholine receptor, infer that then this acetylcholine receptor is the acetylcholine receptor of α 9 α 10 hypotypes.

Each the described polypeptide of the present invention that relates in one aspect to again of the present invention is for the purposes of blockage of acetylcholine receptor; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

Each the described polypeptide of the present invention that relates in one aspect to again of the present invention is in the medicine of preparation blockage of acetylcholine receptor or the purposes in the reagent; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

Each the described polypeptide of the present invention that relates in one aspect to again of the present invention is at preparation treatment or prevention nervous system disorders such as neurodynia, mammary cancer, lung cancer, habituation, parkinsonism or dull-witted etc. medicine, perhaps for the preparation of the purposes of the medicine of kill pests, analgesia, smoking cessation or drug rehabilitation; Particularly, described neurodynia is caused by following reason: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation are hindered, acquired immune deficiency syndrome (AIDS), the head nerves paralysis, drug intoxication, industrial pollution is poisoned, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic congenital sensory neuropathy, acute violent idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, the chronic respiratory obstacle, the composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, the Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

Of the present inventionly relate in one aspect to again a kind of nervous system disorders such as neurodynia, mammary cancer, lung cancer, habituation, parkinsonism or dull-witted etc. method for the treatment of and/or preventing, the perhaps method of a kind of kill pests, analgesia, smoking cessation or drug rehabilitation comprises the polypeptide of the present invention (conotoxin peptide or its propetide) that gives significant quantity or the step of pharmaceutical composition of the present invention; Particularly, described neurodynia is caused by following reason: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation are hindered, acquired immune deficiency syndrome (AIDS), the head nerves paralysis, drug intoxication, industrial pollution is poisoned, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic congenital sensory neuropathy, acute violent idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, the chronic respiratory obstacle, the composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, the Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

Conotoxin peptide of the present invention can by playing a role in conjunction with α 9 α, 10 acetylcholine receptors (nAChR), have analgesic activities.Can be applicable to study, diagnose and treat the nervous system disorders such as neurodynia, mammary cancer, lung cancer, parkinsonism, dementia, habituation and be used for the aspect such as research as useful molecular probe.Different α class CTX is different to the affinity of vertebrates acceptor, sometimes differs several orders of magnitude.Difference between this all system can be used as the different subtype that molecular probe is determined nAchR so that α class CTX can be used as the kind system generation that useful probe is used for research vertebrates nAChR.They are drug candidate, lead drug and the medicine of new drug development.

The below has provided the explanation of the term that the present invention relates to.

Neurodynia

Polypeptide of the present invention relates to the various neuralgic purposes for the treatment of.Neurodynia be around or the pain that causes of former of central nervous system or secondary lesion or dysfunction or of short duration disorder, show as spontaneous pain, allodynia, hyperpathia etc.A lot of diseases all can cause neurodynia, comprise cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation are hindered, acquired immune deficiency syndrome (AIDS), the head nerves paralysis, drug intoxication, industrial pollution is poisoned, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic congenital sensory neuropathy, acute violent idiopathic neuralgia, extruding neurodynia, vasculitis (vasculitis)/local asphyxia, uremia, children's bile hepatic diseases, the chronic respiratory obstacle, the composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, the Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

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 be operatively connected with it, described regulating and controlling sequence can instruct encoding sequence to express in the appropriate 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.

The nucleotide sequence of the separation of operate coding peptide of the present invention makes it express described peptide in many ways.May expect or must before insertion vector, process nucleotide sequence that this depends on expression vector.The technology of using recombinant DNA method modification of nucleic acids sequence is known in the art.

Herein term " regulating and controlling 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 nucleotide sequence of coded polypeptide.These regulating and controlling sequences include, but not limited to leader sequence, Polyadenylation 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.In order to the coding region of regulating and controlling sequence with the nucleotide sequence of coded polypeptide is connected, can provide the regulating and controlling sequence of belt lacing in order to import specific restriction site.Term " is operatively connected " and is defined as in the text a kind of like this conformation, 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 the expression of polypeptide.

Regulating and controlling sequence can be suitable promoter sequence, can be expressed the nucleotide sequence of the host cell identification of nucleotide sequence.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 the born of the same parents of own coding and host cell homology or allos.

Regulating and controlling sequence can also be suitable transcription termination sequence, thereby can be identified one section sequence that termination is transcribed by host cell.Terminator sequence is operatively connected 3 ' end at the nucleotide sequence of coded polypeptide.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, i.e. the mRNA non-translational region very important to the translation of host cell.Leader sequence is operatively connected 5 ' end in the nucleotide sequence of coded polypeptide.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 signal peptide coding region, and encoding one section and be connected in the N-terminal aminoacid sequence of polypeptide in this district, can guide coded polypeptide to enter the emiocytosis approach.5 ' the end in nucleic acid sequence encoding district may be natural contain the translation frame as one man with the signal peptide coding region of the coding region fragment Nature Link 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 contain signal peptide coding region under normal circumstances, may need to add the extraneous signal peptide-coding region.Perhaps, can replace simply natural signal peptide coding region 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 ripe active polypeptide from propolypeptide cutting peptide is former.

When the N-terminal of polypeptide namely 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 in 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, nucleotide sequence and the regulating and controlling sequence of coded polypeptide 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 to prepare recombinant expression vector, this carrier can comprise 1 or a plurality of easily restriction site, in order to insert or replace the nucleotide sequence of coded polypeptide in these sites.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 in order to 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 (namely be present in extrachromosomal complete structure, can be independent of karyomit(e) and copy), 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 copies with the karyomit(e) that is incorporated into.In addition, can use single carrier or plasmid, or totally comprise and will import 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 copied in target host cell.Replication orgin can be with making 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 gene copy number increase of this nucleotide sequence can be by at least 1 additional copies Insertion Into Host Cell genome with this sequence, 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.

Be used for connecting operation that above-mentioned each element makes up recombinant expression vector of the present invention and be well-known to those skilled in the art (referring to such as Sambrook etc., molecular cloning laboratory manual, second edition, 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 occurs between any because replicative phase and the offspring different from 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 the method that restructuring prepares peptide of the present invention, the 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 coding for said peptides; (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; under the condition that allows expression of polypeptides and/or separation, come culturing cell by shake-flask culture, laboratory or industrial fermentation tank 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 produces 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), HPLC, electrophoresis (for example, the isoelectric focusing of preparation property), differential solubleness (for example ammonium sulfate precipitation), SDS-PAGE or extracting is (referring to for example, protein purification, J.C.Janson and Lars Ryden compile, VCH Publishers, New York, 1989).

Transgenic animals and plants

The invention still further relates to the animal or plant cell that has transformed nucleotide sequence of the present invention, the vegetable cells such as preferably wheat, corn are given and are converted the new proterties of host (such as insect-resistance).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 Control pests

Can by the several different methods that those skilled in the art will know that, realize Control pests with conotoxin peptide of the present invention or polynucleotide.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.

Can be with the formulation application of recombinant microorganism that contains conotoxin peptide or comprise polynucleotide of the present invention of preparation in 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 process 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 etc. 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 with at least 1% (weighing scale), and may be 100% (weighing scale).Drying agent has the sterilant of about 1-95% (weighing scale) 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, etc., can be with formulation application in 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 studying, diagnose, alleviate or treatment is relevant with neurodynia, mammary cancer, lung cancer, amentia, habituation, pain, parkinsonism, psychosis, depression, myasthenia gravis etc. disease 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 by the consideration decision of these aspects.

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

The invention still further relates to the pharmaceutical composition of specific blockage nAChRs.

Can use conotoxin peptide of the present invention to occur be used to zoologizeing the kind system of nAChR as useful probe; Determine the different subtype of nAChR as molecular probe; As molecular model, the design new drug; Instrument medicine and medicine as research, diagnosis nervous system disease such as Parkinson's disease, action obstacle, schizophrenia etc.; The candidate medicine for the treatment of mammary cancer, lung cancer, small cell lung cancer etc.As the polypeptide sterilant, be developed as novel biopesticide etc.

The beneficial effect of the invention

α B-conotoxin peptide of the present invention is blockage of acetylcholine receptor (nAChRs) specifically, and the effect of tool analgesic activities and inhibition mammary cancer and lung cancer cell growth.

Description of drawings

Fig. 1: the classification of conotoxin (peptide) and the molecular target of effect thereof.

Fig. 2: demonstration be α B-VxXXIIIA mature peptide sequence (SEQ ID NO:1) and isomer with 3 kinds of possible disulfide linkage mode of connection.Wherein the disulfide linkage mode of connection of VxXXIIIA12 is I-II, III-IV; The disulfide linkage mode of connection of VxXXIIIA13 is I-III, II-IV; The disulfide linkage mode of connection of VxXXIIIA14 is I-IV, II-III.

What Fig. 3: A showed is that 10 μ M α B-VxXXIIIA12 are to the current affects situation of α 9 α 10nAChR.The contrast electric current that " Control " refers among the figure, arrow refer to 10 μ M α B-VxXXIIIA12 incubations 5 minutes, and First Pulse is the current locus (～0 μ A) of first Ach pulse formation behind the incubation.B shows be 3 isomer of α B-VxXXIIIA to the concentration dose response curve of α 9 α 10nAChR, X-coordinate is the logarithmic value (Log[Toxin Concentration] M) of the volumetric molar concentration (M) of used α B-VxXXIIIA isomer among the figure; Ordinate zou is dose response percentage ratio (%Response), is the ratio percentage ratio of acetylcholine receptor electric current and contrast electric current under the detoxifying function of respective concentration.

Fig. 4: demonstration be 10 μ M α B-VxXXIIIA12 to α 9 α 10, α 7, α 3 β 4 are with the current affects situation of α 4 β 2nAChRs.The contrast electric current that " C " refers among the figure, what be right after " C " back is the current locus that α B-VxXXIIIA12 blocks first Ach pulse formation of corresponding receptor subtype.10 μ M α B-VxXXIIIA12 specific blockage α, 9 α 10nAChR, and do not block α 7 fully, α 3 β 4, α 4 β 2nAChRs hypotypes.

Fig. 5: demonstration be with barium ion ND96 perfusate (Ba ⁺⁺-when ND96) replacing conventional ND96 perfusate, α B-VxXXIIIA12 is to the concentration dose response curve of α 9 α 10nAChR, and X-coordinate is the logarithmic value (Log[Toxin Concentration] M) of the volumetric molar concentration (M) of used 3 α B-VxXXI I IA isomer among the figure; Ordinate zou is dose response percentage ratio (%Response), is the ratio percentage ratio of acetylcholine receptor electric current and contrast electric current under the detoxifying function of respective concentration.VxXXIIIA12 is the strongest to the blocking-up activity of α 9 α 10nAChR.With etc. the barium ion ND96 perfusate of volumetric molar concentration replace calcium ion (Ca in the conventional ND96 perfusate ⁺⁺), can effectively stop Xenopus Oocytes to activate by flow of calcium ions and produce chlorion (Cl ^-) the outflow electric current.

Embodiment

Below in conjunction with embodiment embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples only are used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment, according to the described technology of the document in this area or condition (such as works such as reference J. Pehanorm Brookers, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press), corresponding reference or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

Embodiment 1: the extraction of the total RNA of cone shell poison pipe and the structure of cDNA library thereof

From Hainan Island, the Xisha Islands is coastal collects calamus cone shell (Conus vexillum); total RNA with the total RNA extraction agent of the centrifugal tissue/cell of a small amount of post box (Shanghai China Shun biotechnology company limited) extraction calamus cone shell poison pipe operates according to the test kit specification sheets.Total RNA with extracting scans under 200～300nm wavelength with PE company nucleic acid-protein analyser, detects content and the purity of RNA; Detect the integrity of RNA with 1.1% agarose gel electrophoresis.

Press the Creator of Clontech company ^TMSMART ^TMCDNA library makes up the test kit specification sheets and makes up calamus cone shell poison pipe cDNA library, is summarized as follows: get the total RNA of 3 μ L (about 1.0 μ g), add CDSIII/3 ' PCR Primer (1 μ L), SMARTIV ^TMOligonucleotide (1 μ L), 72 ℃ of incubation 2min.Add again 5 * First-Strand Buffer (2.0 μ L), 1.0 μ L dNTPMix (10mmol/L), 1 μ L DTT (20mmol/L), PowerScript ThermoScript II (1 μ L).42 ℃ of temperature are bathed synthetic cDNA the first chain of 1h.Take cDNA the first chain as template, take CDSIII/3 ' PCR primer and 5 ' PCR as primer, with LD-PCR (long-distance PCR) synthetic double chain cDNA.Reaction conditions: 95 ℃ of 20s, 95 ℃ of 5s, 68 ℃ of 6min, the agarose electrophoresis with 1.1% detects the synthetic quality of double-stranded cDNA.Behind the PCR product purification, cut through the SfiI enzyme, carry out fractional separation at CHROMA SPIN-400 post; Every pipe is got 3 μ L with the size distribution of 1.1% agarose gel electrophoresis detection cDNA after separating, and the collection tube of cDNA fragment greater than 500bp merged, and is resuspended for subsequent use with 7 μ L deionized waters behind the purifying.

Get the double-stranded cDNA of 1.5 μ L and 1.0 μ L pDNR-LIB (0.1 μ g/mL, the carrier that SfiI processed), 0.5 μ L, 10 * Ligation Buffer, 0.5 μ L ATP (10mmol/L), 0.5 μ L T4DNA Ligase, deionized water 1 μ L, 16 ℃ of connections are spent the night.After connecting product purification, with electroporation recombinant plasmid is imported competent cell intestinal bacteria JM-109 ^[8]Compile satisfactory transformation mixture, thereby form original cDNA library, after amplification, obtain satisfactory cDNA library.

Embodiment 2: the new superfamily conotoxin gene cloning of α B-, separation and sequential analysis

(Kaas, Q. that the conotoxin mature peptide is normally formed after posttranslational modification is sheared by its precursor protein; Yu, R.; Jin, A.H.; Dutertre, S.; Craik, D.J., ConoServer:updated content, knowledge, and discovery tools in the conopeptide database.Nucleic acids research 2012,40 (Database issue), D325-30) the signal peptide high conservative of the conotoxin of same superfamily, and have identical halfcystine pattern.This conservative property provides effective way (Zhangsun, D. for a certain specific superfamily conotoxin newcomer's discovery and evaluation; Luo, S.; Wu, Y.; Zhu, X.; Hu, Y.; Xie, L., Novel O-superfamily conotoxins identified by cDNA cloning from three vermivorous Conus species.Chemical biology ﹠amp; Drug design 2006,68 (5), 256-65.).

The present invention from calamus cone shell poison pipe cDNA library at random the mono-clonal of about 50 of pickings carry out sequencing analysis, institute's cDNA sequence that obtains is through the DNAStar software analysis, knows its open reading frame (ORF) sequence, coded protein sequence, 3 '-and 5 '-non-translational region (UTR) sequence.The prediction of the signal peptide of conotoxin precursor protein, propetide and mature peptide adopts online ProP 1.0Server to analyze (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites.Protein engineering, design ﹠amp; Selection:PEDS 2004,17 (1), 107-12.).We have therefrom found the cDNA sequence (SEQ ID NO:3) of a uniqueness, contain 1 open reading frame (SEQ ID NO:4), the propeptide sequence (SEQ ID NO:2) that its coding produces only contains signal peptide and two integral parts of mature peptide (SEQ ID NO:1), this with all conotoxin precursor peptides of in the past finding by signal peptide, propetide, 3 integral parts of mature peptide are all different, have unique 4 halfcystine patterns " C-CC-C ", this halfcystine pattern from all conotoxins of finding in the past is all different, signal peptide sequence is all different from the signal peptide of known superfamily, thereby this SEQ ID NO:3cDNA coding is a new superfamily conotoxin, our called after B-superfamily, the 23rd (XXIII) plants the halfcystine pattern, and is the conotoxin of first this superfamily of identifying from the calamus cone shell.

Follow-up study shows that it is the nAChRs blocker, so definite designation is α B-conotoxin VxXXIIIA, is abbreviated as α B-VxXXIIIA or VxXXIIIA.Corresponding sequence and number as follows.

Mature peptide aminoacid sequence shown in the SEQ ID NO:1 (hereinafter being also referred to as α B-conotoxin VxXXIIIA or α B-VxXXIIIA or VxXXIIIA): VRCLEKSGAQPNKLFRPPCCQKGPSFARHSRCVYYTQSRE.

Precursor peptide aminoacid sequence shown in the SEQ ID NO:2 (hereinafter being also referred to as α B-conotoxin VxXXIIIA precursor or α B-VxXXIIIA precursor or VxXXIIIA precursor): METLTLLWRASSSCLLVVLSHSLLRLLGVRCLEKSGAQPN KLFRPP CCQKGPSFARHSRCVYYTQSRE.

The full-length cDNA polynucleotide sequence of the VxXXIIIA precursor protein shown in the SEQ ID NO:3: GACTCCTATTGTCATA AtgGAGACTCTCACTCTACTATGGCGAGCGTCGTCGTCTTG CCTGCTCGTCGTTCTCAGTCACTCGCTTCTTCGTCTTCTCGGGGTTAGGTGTTTGG AGAAG AGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTGTCAAAAGGGCCCTTC TTTCG CCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAGtaaTCCGGGGGGTCCGAAATGTGAAATAAGAAGGCCGAGCATACAACCCTCCTTAACACTCGCCTATTGTAAAAGGGTGTCCTTTGTCAATAGTGGTACTAATGTTGTTTAAAAAATCCGCTGTGATATCTTAGGTGTTCAAGTAGTAGGTTGCACCCCCCTCGAGAGGGTATACCAGCTGGTCGTCCGCCGGCTATTAAGTGATCACTCACCCTTTGTTGATGGTGGTACAAGTTAAAGGATCCCCCGGGCGCGTGGTTTTCTTACCACATTTTTAGAAGTCCCCCCGGCGCCGCCGGTTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

Open reading frame (ORF) polynucleotide sequence of the coding VxXXIIIA precursor protein shown in the SEQ ID NO:4: AtgGAGACTCTCACTCTACTATGGCGAGCGTC GTCGTCTTGCCTGCTCGTCGTTCTCAGTCACTCGCTTCTTCGTCTTCTCGGGGTTA GGTGT TTGGAGAAGAGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTGTCAAAA GGGCC CTTCTTTCGCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAGtaa

The polynucleotide sequence of the coding VxXXIIIA mature peptide shown in the SEQ ID NO:5: GTT AGGTGTTTGGAGAAGAGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTG TCAAA AGGGCCCTTCTTTCGCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAG taa

Embodiment 3: the synthetic of α B-conotoxin VxXXIIIA

According to the aminoacid sequence (SEQ ID NO:1) of α B-conotoxin VxXXIIIA mature peptide, adopt three kinds of possible isomer linear peptides VxXXIIIA12 of Fmoc method synthetic VxXXIIIA, VxXXIIIA13, VxXXIIIA14 (Fig. 2).Concrete grammar is as follows.

The resin peptide of three isomer adopts the Fmoc chemical process to carry out synthetic, except halfcystine, and all the other amino acid side chain protected group of standard.The the 1st and the 2nd halfcystine (Cys) of VxXXIIIA12-SH Trt (S-trityl) protection, the 3rd and the 4th halfcystine-SH in pairs protection of Acm (S-acetamidomethyl); The the 1st and the 3rd halfcystine (Cys) of VxXXIIIA13-SH Tr t (S-trityl) protection, the 2nd and the 4th halfcystine-SH in pairs protection of Acm (S-acetamidomethyl); The the 2nd and the 3rd halfcystine (Cys) of VxXXIIIA14-SH Trt (S-trityl) protection, the 1st and the 4th halfcystine-SH in pairs protection of Acm (S-acetamidomethyl).Its synthesis step is: adopt Fmoc and FastMoc method in the solid-phase synthesis, synthesized 3 isomer linear peptides at ABI Prism 433a Peptide synthesizer.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 amidated resin and Fmoc amino acid, the synthetic handbook of synthesis step reference instrument carries out.For reacting completely, at piperidines deprotection and coupling time difference proper extension, difficulty is connect amino acid adopt two couplings, obtain resin peptide.With reagent K (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole; 90: 5: 2.5: 7.5: 5, v/v/v/v/v) linear peptides is cut down from resin, and with icing ether sedimentation and washing and recycling linear peptides crude product, with preparation type reverse hplc C18 post (Vydac) purifying, the wash-out linear gradient is 60%CAN (acetonitrile) for 20-60%B60. solvent B in 0-40min, 40%H20,0.92%TFA (trifluoroacetic acid); Solvent orange 2 A is the aqueous solution of 1%TFA.

Linear peptides behind the purifying carries out purity detecting with the HPLC C18 post (Vydac) of analysis mode, and gradient is 0-50min 0-50%buffer B, and 50-55min 50-100%bufferB, flow velocity are 1mL/min.Its purity reaches more than 95%, is used for oxidative folding.

Reference literature (Dowell, C.; Olivera, B.M.; Garrett, J.E.; Staheli, S.T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J.M.; McIntosh, J.M., Alpha-conotoxin PIA is selective for alpha6subunit-containing nicotinic acetylcholine receptors.The Journal of neuroscience 2003,23 (24), 8445-52.) linear peptides of 3 isomer VxXXIIIA12, VxXXIIIA13 and VxXXIIIA14 is carried out the folding reaction of two-step oxidation, process is summarized as follows:

At first (pH 7.5 for 20mM potassium ferricyanide, 0.1M Tris, 30min) form first pair of disulfide linkage between two halfcystines of Trt blocking group by Tripotassium iron hexacyanide oxidation style.Monocyclic peptide is carried out iodine oxidation (10mM iodine in H behind reversed-phase HPLC C18 post (Vydac) purifying ₂O:trifluoroacetic acid:acetonitrile (78: 2: 20by volume, 10min) removes the Acm on other 2 halfcystines, forms simultaneously second pair of disulfide linkage between these 2 halfcystines.Two cyclic peptide namely obtain according to order directed α B-conotoxin that forms disulfide linkage between corresponding halfcystine of holding from N to the C end again through reversed-phase HPLC C18 post (Vydac) purifying, and are accredited as correctly by mass spectrum (MS).

The theoretical molecular of 3 isomer behind the oxidative folding (monoisotopic mass) is 4622.27Da, and the determining molecular weight of VxXXIIIA12 is 4622.3Da; The determining molecular weight of VxXXIIIA13 is 4622.2Da; The determining molecular weight of VxXXIIIA14 is 4622.4Da.Peptide concentration calculates peptide concentration and quality with colorimetric estimation under the 280nm wavelength according to Beer-Lambert equation (equation).The isomer of these quantitative mistakes continues on for follow-up active testing.

Embodiment 4: α B-conotoxin VxXXIIIA specific blockage α 9 α 10nAChR experiment

Reference literature (Azam L, Yoshikami D, McIntosh JM.Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A, E11A, L15A] .J Biol Chem.2008; 283 (17): the method 11625-32.), and in-vitro transcription test kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) specification sheets, prepare various rat nervous system type nAChRs hypotype (α 3 β 2, α 6/ α 3 β 2 β 3, α 6/ α 3 β 4, α 9 α 10, α 4 β 2, α 4 β 4, α 3 β 4, α 2 β 2, α 2 β 4, α 7) and the cRNA of mouse muscle type nAChRs (α 1 β 1 δ ε), its concentration is calculated with the OD value under the UV 260nm.Dissect and collect Africa xenopus (Xenopus laveis) ovocyte (frog's egg), cRNA is injected in the frog's egg, the injection volume of each subunit is 5ng cRNA.Each subunit injection 0.5-2.5ng DNA of muscle nAChR.Frog's egg is cultivated in ND-96.Injection cRNA in 1-2 days after frog's egg is collected, injection is used for the voltage clamp record of nAChRs in rear 1-4 days.

1 frog's egg of injecting cRNA is placed the Sylgard track (diameter 4mm * degree of depth 2mm) of 30uL, gravity perfusion contains ND96 perfusate (the 96.0mM NaCl of 0.1mg/ml BSA (bovine serum albumin), 2.0mM KCl, 1.8mM CaCl ₂, 1.0mM MgCl ₂, 5mM HEPES, pH 7.1-7.5) or contain the ND96 (ND96A) of 1mM atropine, flow velocity is 1ml/min.All conotoxin solution also contains 0.1mg/ml BSA to reduce the non-specific adsorption of toxin, with transforming valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) can between perfusion toxin or vagusstoff (ACh), carry out free switching, and a series of threeway solenoid valve (solenoid valves, model161TO31, Neptune Research, Northboro, MA) make between perfusion ND96 and the ACh etc. and carry out free switching.The electric current of Ach gate is arranged on " slowly " pincers by two electrodes voltage clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT), and clamp gain carries out online record when maximum value (* 2000) position.With glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) the drawn glass electrode of 1mm external diameter * 0.75 internal diameter mm, and be full of 3MKCl as the voltage and current electrode.The membrane voltage strangulation in-70mV. whole system by computer control and record data.The ACh pulse is the ACh every 5mi n automatic filling 1s.The concentration of ACh is respectively, and nAChRs and the nervous system type α 9 α 10nAChRs ovum of expressing muscularity are 10 μ M; The α 7 that expresses the nAChRs of nervous system type is 200 μ M, and other hypotype all is 100 μ M.At least record 4 ovum and express certain hypotype to the current response situation of different toxin concentrations, and current locus.

The current data of test is carried out statistical study with GraphPad Prism software (San Diego, CA), draws dose response curve, calculates the various parameters of the multiple relevant toxin blocking-up nAChRs such as hemiblock concentration IC50 of conotoxin.

The result shows that 10 μ M α B-VxXXIIIA12 (embodiment 3 preparations) have almost completely blocked the open electric current that produces by the α 9 α 10nAChR of Ach gate, and wash-out is very fast, and blocking-up is reversible (Fig. 3 A).In 3 isomer, the activity of α B-VxXXIIIA12 is the strongest, and the activity of α B-VxXXIIIA13 is taken second place, and α B-VxXXIIIA14 does not almost have activity (Fig. 3 B).They partly block dosage IC50 and limit of error is respectively α B-VxXXIIIA12,1.2 μ M (0.8-1.7 μ M); α B-VxXXIIIA13,3.9 μ M (2.7-5.6 μ M); Slope (Hillslope) and the limit of error of α B-VxXXIIIA14＞30 their dose response curves of μ M. are respectively VxXXIIIA12,1.4 (0.5-2.1) and VxXXIIIA13,1.3 (0.9-1.7). therefore, follow-up blocking-up for other nAChRs hypotypes is active, just only detects with the strongest active α B-VxXXIIIA12 isomer.α B-VxXXIIIA12 is as shown in table 1 to the slope of partly blocking dosage IC50 and dose response curve of various nAChRs hypotypes.

Table 1: α B-VxXXIIIA12 is to the slope of partly blocking dosage IC50 and dose response curve of various nAChRs hypotypes

Annotate: a is that degree of confidence is 95% interval.

α B-VxXXIIIA12 is high to the blocking-up selectivity of α 9 α 10nAChR.From 10 μ M α B-VxXXIIIA12 to α 9 α 10, α 7, α 3 β 4, can find out (Fig. 4) with the current affects situation of α 4 β 2nAChRs, α B-VxXXIIIA12 specific blockage α 9 α 10nAChR, and the toxin of same concentrations is to α 7, and α 3 β 4 are active without any blocking-up with α 4 β 2nAChRs hypotypes.

Known α 9 α 10nAChR are to calcium ion (Ca ⁺⁺) have a very high permeability.Flow of calcium ions by nAChRs can activate generation chlorion (Cl ^-) the outflow electric current, on Xenopus Oocytes, this electric current accounts for open more than 90% of electric current of the α 9 α 10nAChR that observe.On the contrary, the barium ion (Ba approaching with calcium ion ⁺⁺) can not activate and produce the chlorion electric current.Thereby we use barium ion ND96 perfusate (Ba ⁺⁺-ND96,1.8mM BaCl ₂Replace CaCl ₂) replacing conventional ND96 perfusate, the open current ratio of the α 9 α 10nAChR that observe is little much with the electric current of conventional ND96 perfusate, and this is consistent with research in the past.Under barium ion ND96 perfusate condition, VxXXIIIA12 is the strongest to the blocking-up activity of α 9 α 10nAChR, and VxXXIIIA13 takes second place, and VxXXIIIA14 does not have activity (Fig. 5).They partly block dosage I C50 and limit of error is respectively α B-VxXXIIIA12,1.5M (1.2-1.9); α B-VxXXIIIA13,3.1M (1.9-4.9).The slope of their dose response curve (Hillslope) and limit of error are respectively VxXXIIIA12,0.77 (0.62-0.93) and VxXXIIIA13,0.77 (0.43-0.89). under barium ion ND96 perfusate condition, the activity of 3 isomer of α B-VxXXIIIA is to similar in the result who contains under the normal ND96 perfusate condition of calcium ion.Therefore, VxXXIIIA has blocked α 9 α 10nAChR really, rather than blocking-up is because of calcium ion activated chlorion electric current.

Studies show that α 9 α 10nAChR are novel targets (McIntosh, J.M. for the treatment of neurodynia, cancer chemotherapy, mammary cancer, lung cancer, wound healing etc.; Absalom, N.; Chebib, M.; Elgoyhen, A.B.; Vincler, M., Alpha9nicotinic acetylcholine receptors and the treatment of pain.Biochemical pharmacology2009,78 (7), 693-702.Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vcl.1alleviates neuropathic pain and accelerates functional recovery of injured neurones.Brain research 2005,1059 (2), 149-58.Holtman, J.R.; Dwoskin, L.P.; Dowell, C.; Wala, E.P.; Zhang, Z.; Crooks, P.A.; McIntosh, J.M., The novel small molecule alpha9alpha10nicotinic acetylcholine receptor antagonist ZZ-204Gis analgesic.European journal of pharmacology 2011,670 (2-3), 500-8.Zheng, G.; Zhang, Z.; Dowell, C.; Wala, E.; Dwoskin, L.P.; Holtman, J.R.; McIntosh, J.M.; Crooks, P.A., Discovery of non-peptide, small molecule antagonists of alpha9alpha10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain.Bioorganic ﹠amp; Medicinal chemistry letters 2011,21 (8), 2476-9.Chernyavsky, A.I.; Arredondo, J.; Vetter, D.E.; Grando, S.A., Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization.Experimental cell research 2007,313 (16), 3542-55; Chikova, A.; Grando, S.A., Naturally occurring variants of human Alpha9nicotinic receptordifferentially affect bronchial cell proliferation and transformation.PloS one 2011,6 (11), e27978.).Therefore, the new superfamily conotoxin VxXXIIIA of α B-of the present invention has high using value aspect the mechanism research of above-mentioned disease, diagnosis, the treatment.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Claims

1. a peptide species, its for or comprise and be selected from each described aminoacid sequence in following (1) to (3):

(1) aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:2;

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

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

2. polypeptide according to claim 1, wherein, first halfcystine of the N-terminal among the SEQ ID NO:1 and second halfcystine form disulfide linkage, and the 3rd halfcystine and the 4th halfcystine formation disulfide linkage; Perhaps first halfcystine and the 3rd halfcystine form disulfide linkage, and second halfcystine and the 4th halfcystine formation disulfide linkage; Perhaps first halfcystine and the 4th halfcystine form disulfide linkage, and second halfcystine and the 3rd halfcystine formation disulfide linkage.

3. polynucleotide, the aminoacid sequence of its coding claim 1 or 2 described polypeptide.

4. polynucleotide according to claim 3, its for or comprise and be selected from each described nucleotide sequence in following (1) to (3):

(1) nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4 or the SEQ ID NO:5;

(2) complementary sequence of the complementary sequence of the complementary sequence of SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5;

(3) under rigorous condition can with the nucleotide sequence of the nucleotide sequence hybridization described in above-mentioned (1).

5. nucleic acid construct, it comprises claim 3 or 4 described polynucleotide.

6. expression vector, it comprises nucleic acid construct claimed in claim 5.

7. the cell of a conversion, it comprises expression vector claimed in claim 6.

8. fusion rotein, it comprises claim 1 or 2 described polypeptide.

9. pharmaceutical composition, it comprises claim 1 or described polypeptide, perhaps comprises fusion rotein claimed in claim 8; Alternatively, it also comprises pharmaceutically acceptable carrier or auxiliary material.

One kind in vivo or extracorporeal blocking acetylcholine receptor or the method for regulating levels of acetylcholine, comprise the claim 1 of using significant quantity or the step of 2 described polypeptide; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

11. a method of screening acetylcholine receptor inhibitor or definite acetylcholine receptor subtypes, the method comprises: by the step that in the situation that has and do not exist the candidate compound existence acetylcholine receptor is contacted with claim 1 or 2 described polypeptide; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

12. claim 1 or 2 described polypeptide are used for the purposes of blockage of acetylcholine receptor; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

13. claim 1 or 2 described polypeptide are in the medicine of preparation blockage of acetylcholine receptor or the purposes in the reagent; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptors.

14. claim 1 or 2 described polypeptide treat and/or prevent for example purposes of neurodynia, cancer chemotherapy, mammary cancer, lung cancer, wound healing, habituation, parkinsonism or dull-witted medicine of nervous system disorders in preparation, perhaps for the preparation of the purposes of the medicine of kill pests, analgesia, smoking cessation or drug rehabilitation; Particularly, described neurodynia is caused by following reason: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation are hindered, acquired immune deficiency syndrome (AIDS), the head nerves paralysis, drug intoxication, industrial pollution is poisoned, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic congenital sensory neuropathy, acute violent idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, the chronic respiratory obstacle, the composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, the Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, or allergy.

15. the preparation method of claim 1 or 2 described polypeptide comprises the steps: