CN103374066B - Alpha B-O-conotoxin and medicine composition and application thereof - Google Patents

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 the propetide of described conotoxin peptide, its nucleic acid construct, the cell of its expression vector and conversion and its 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, it mediates the physiological function of numerous maincenter and peripheral nervous system, comprises study, memory, response, 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 above-mentioned disease also be there is no to the medicine of symptomatic treatment.Conventional nonselective nAChR agonist is as nicotine, although can alleviate the symptom of above-mentioned sacred disease, they produce strong side effect to heart and gi tract, and have additive.Therefore, the ligand drug that exploitation has highly selective for the various hypotype of nAChRs is key point (the Livett BG treating above-mentioned disease, 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).But the prerequisite will developing such medicine is, obtain can the alternative cpd of the various hypotype of specific combination nAChRs, studies and identify meticulous composition and the physiological function of various hypotype as instrument medicine, or directly as the medicine of relative disease.In addition, in mammary cancer and small cell lung cancer, on tumor cell membrane, the activation of nAChR promotes tumor cell proliferation, with the activation of these acceptors of drugs block, effectively can carry out early diagnosis, or treat these calamitous cancers.

NAChRs is assembled into a variety of hypotype by different α and β subunits, and often kind of hypotype has distinct pharmacological characteristic.Owing to lacking the highly selective ligand compound for various hypotype, fine structure and the function that study and illustrate various nAChRs hypotype face lot of challenges.

Research shows, α 9 α 10nAChR is novel targets (McIntosh, the 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 blocker has treatment neurodynia and accelerates the function that injured nerve recovers, and may be to be played a role (Holtman, J.R. by immunologic mechanism, 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 & medicinal chemistry letters 2011, 21 (8), α 9 α 10nAChR 2476-9.) on keratinocyte plays a part very important (Chernyavsky in the pathophysiological processes of wound healing, A.I., 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 research shows, α 9nAChR subunit process LAN in breast cancer tissue.α 9 subunit variant affects conversion and the propagation of bronchial epithelial cells, this subunit has 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 caused by the nicotine (Nicotine) in tobacco, 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 (such as respiratory system, the recycle system, neural system, urinary system and other important organ), and the special harm to pregnant woman and fetus.According to investigations, smoking is the first killer of American's health, and every year because dead number of smoking is up to people more than 430,000, shelter has first of the cause of death, be secondly the death toll that alcoholism causes is annual about 100,000 people.Smoke general especially in China, and harm is more serious, have every day 2000 Chinese to be dead because of smoking, to the year two thousand fifty, this death toll will rise to people's every day 8000.The cause of the death that smoking causes is mainly from lung cancer and tuberculosis, and the medicine treating 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 toponarcosis medication, blocks the pain signal because peripheral nerve, neuroplexus, Dorsal ganglion, sympathetic nervous system etc. produce.But these treatments can only have analgesic effect the short period of time, but can not effect a radical cure neurodynia.A lot of disease all can cause neurodynia, comprise cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation wound, acquired immune deficiency syndrome (AIDS), head nerves is paralysed, drug intoxication, industrial pollution is poisoning, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic idiopathic esthesioneurosis, acute severe idiopathic neuralgia, extruding neurodynia, vasculitis (vasculitis)/local asphyxia, uremia, children's bile hepatic diseases, chronic respiratory failure, composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

Analgesic effect (Vincler is played by intramuscular injection with the neuralgia medicine that α 9 α 10nAChR is target spot, 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 current business-like ω-CTX MVIIA analgesic-Qi Kaonuo peptide route of administration, Qi Kaonuo peptide need be built in human body by program pump and be administered directly to spinal cord, route of administration is pretty troublesome, this administration pump is very expensive, be only limitted to developed country's application such as USA and Europe at present, 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, analgesic activity more powerful and can the s-generation CTX new drug of intramuscular injection, will more wide market be had.

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

Conotoxin (conopeptide, conotoxin, CTX) is made up of 7-50 amino-acid residue mostly, be rich in the neuropeptide toxin of halfcystine (Cys).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 family such as α, ω, μ, δ by its acceptor target position.Each superfamily is according to acceptor target type, α, α A, κ A (A-superfamily) can be divided into again, ω, δ, κ, μ O (O-superfamily), μ, φ, KM (M-superfamily) etc. family (hypotype) (Fig. 1).

Conotoxin first synthesizes larger non-activity polypeptide precursor in vivo, they are made up of 50-80 amino-acid residue, generally by signal peptide, N-end or C-CICP and mature peptide 3 part composition, these signal peptide sequences have conservative property in all superfamily members, proteolysis signal often containing standard between propetide and mature peptide, as basic amino acids lysine (K) or arginine (R) etc., mature peptide district is except halfcystine, its sequence has highly variable, thus cause respective action target spot and pharmacological activity difference far away.Propetide district is conducive to secretion (Hidaka, the Y. that mature peptide oxidative folding forms correct disulfide linkage or is conducive to 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.).

Visible, the novel blocker of nAChR particularly α 9 α 10nAChR has important using value for the research of this receptor structure and fuction, and is likely developed as the original medicine of the closely related disease with this receptor.Therefore, the nAChRs blocker finding new high specific is needed badly.

Summary of the invention

The present inventor is through deep research and creatively work, and has found the new superfamily conotoxin peptide of a class α B-.The new superfamily conotoxin peptide of α B-of the present invention has 4 unique halfcystine patterns " C-CC-C ", and not containing propetide district, these are all different with precursor peptide compositing area from the halfcystine pattern of all conotoxins found in the past.The present inventor is surprised to find, it can blockage of acetylcholine receptor specifically, particularly the strongest to the blocking-up activity of neurodynia drug target, mammary cancer, lung cancer target spot α 9 α 10nAChR, have preparing analgesic, about the applications well prospect in cancer treatment drugs and neuroscience instrument medicine etc.Thus provide following invention:

One aspect of the present invention relates to a peptide species, its for or comprise the aminoacid sequence be selected from according to any one of following (1) to (4):

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

(2) aminoacid sequence (precursor peptide) shown in SEQ ID NO:2 (hereinafter 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%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, most preferably at least 97% identical aminoacid sequence (being called in this article " homeopeptide "); Or

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

Here is the precursor peptide open reading frame of α B-superfamily conotoxin VxXXIIIA, and the encode precursor peptide (SEQ ID NO:2) and mature peptide (SEQ ID NO:1) that produce.Arrow refer to presumption proteolysis be processed as the site (G) of mature peptide.The precursor peptide of VxXXIIIA is only containing signal peptide and mature peptide 2 regions, and signal peptide region italic indicates, and mature peptide region (SEQ ID NO:1) indicates with underscore, and terminator codon indicates with " * ".It is all different that this and all conotoxins found in the past all hold propetide and mature peptide or C-to hold propetide 3 regions to form structure by signal peptide, N-, it is a new superfamily conotoxin, the new superfamily of called after B-, again due to this toxin blocks 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

In order to 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 [inquiry 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 the cost extending breach,-v refers to that single line describes (one-line description) number,-b refers to the ratio logarithm that will show,-I refers to inquire about document,-d refers to the database for inquiring about.

In the aminoacid sequence of homeopeptide and SEQ ID NO:1-2 arbitrary aminoacid sequence difference may be to have replaced, insert, add and/or lacked 1 or multiple, preferred 1-5, more preferably 1-3, especially preferably 1-2, most preferably 1 amino-acid residue.Preferably, amino acid change is that character changes less change, is namely the folding and/or active conservative amino acid of remarkably influenced protein to replace; Small segment lacks, normally 1 to about 5, preferably 1-3, more preferably 1 amino acid whose disappearance; Little amino or C-terminal extend, as the methionine residues that aminoterminal adds; There is the little connection peptides of nearly about 20-25 residue; Or the little extension contributing to purifying by changing net charge or other function is as polyhistidine fragment, epitope or land.

The example that conservative property replaces is the replacement carried 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 such as H.Neurath and R.L.Hill, 1979, at " protein " book, described in Academic Press, 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 be reversed.

The present invention is also included in the N-end of α B-conotoxin peptide of the present invention and/or C-the terminal fusion fusion polypeptide of other peptide/polypeptide or the fusion polypeptide of cleavable.The technology producing fusion polypeptide is known in the art, comprise the encoding sequence of encoding sequence and coding other peptide/polypeptide described connecting code book invention peptide, make them in same frame, and the expression of fusion polypeptide is controlled by identical promotor and terminator.

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

Polypeptide according to any one of the present invention, wherein, first halfcystine and second halfcystine of the N-terminal in SEQ ID NO:1 form disulfide linkage, and the 3rd halfcystine and the 4th halfcystine form disulfide linkage; Or first halfcystine and the 3rd halfcystine form disulfide linkage, and second halfcystine and the 4th halfcystine form disulfide linkage; Or first halfcystine and the 4th halfcystine form disulfide linkage, and second halfcystine and the 3rd halfcystine form disulfide linkage.

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

Extract the calamus cone shell (Conus C.Vexillum) that above-mentioned conotoxin peptide can produce from China Hainan.Also can the amino acid sequence (method in such as reference example 3) of chemosynthesis; Or express its Nucleotide (the preparation reference example 1-2 of nucleotide sequence or directly according to the nucleotide sequence synthetic in embodiment 2) by the means of gene recombination, obtain polypeptide.Also can with reference to method below:

Another aspect of the present invention relates to the preparation method of the polypeptide described in any one 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 Trt or Acm blocking group, between corresponding halfcystine, fixed point forms disulfide linkage respectively;

2) by 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 preparative reverse hplc C18 post (Vydac) purifying;

3) by step 2) in the product that obtains carry out two-step oxidation and fold.

Another aspect of the invention relates to a kind of polynucleotide, the aminoacid sequence of polypeptide described in its any one of code book invention.

Polynucleotide according to any one of the present invention, its for or comprise the nucleotide sequence be selected from according to any one of following (1) to (4):

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

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

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

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

(5) under high stringency conditions can with the nucleotide sequence of nucleotide sequence hybridization according to any one of above-mentioned (1) to (4).

Here 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 polynucleotide, there is numerous documents can be for reference in the prior art, comprise such as Sambrook etc., Molecular Cloning: A Laboratory room handbook, the second edition, cold spring harbor laboratory, cold spring port, 1989.The high stringency conditions of various degree can be applied, such as moderate, medium-high in hybridization, or high stringent condition.More rigorous condition, the complementarity forming duplex requirement is higher.Temperature, concentration and probe concentration, probe length, ionic strength, time etc. can be passed through and control Stringency.For double-stranded DNA gene probes, hybridize in lower than DNA heterozygote melting temperature (Tm) [melting temperature, Tm]) spend the night in 6X SSPE, 5XDenhardtShi solution, 0.1%SDS, 0.1mg/ml denatured DNA at 20-25 DEG C.Cleaning is carried out usually as follows: in Tm-20 DEG C in 0.2X SSPE, 0.1%SDS one time 15 minutes (moderate stringency conditions cleaning).

Another aspect of the invention relates to one or more primer pairs, the full-length cDNA polynucleotide sequence SEQ ID NO:3 of VxXXIIIA precursor protein is comprised according to it, or the primer pair designed by open reading frame (ORF) polynucleotide sequence SEQ ID NO:4 of coding VxXXIIIA precursor protein.Described primer pair can be used in pcr amplification, obtains SEQ ID NO:3 or the polynucleotide sequence shown in SEQ ID NO:4.

Another aspect of the invention relates to a kind of nucleic acid construct, and it comprises the polynucleotide described in any one of the present invention.

Another aspect of the invention relates to a kind of expression vector, and it comprises the nucleic acid construct described in any one of the present invention.

Another aspect of the invention relates to a kind of cell of conversion, and it comprises the expression vector described in any one of the present invention.

Another aspect of the invention relates to a kind of fusion rotein, and it comprises the polypeptide described in any one of the present invention.

Another aspect of the invention relates to a kind of pharmaceutical composition, and it comprises the polypeptide described in any one of the present invention, or comprises fusion rotein of the present invention; Alternatively, it also comprises pharmaceutically acceptable carrier or auxiliary material.

Another aspect of the invention relates to a kind of method of blockage of acetylcholine receptor, comprises the step of the polypeptide described in any one of the present invention using significant quantity; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.

Another aspect of the invention relates to a kind of method of screening acetylcholine receptor inhibitor or determining acetylcholine receptor subtypes, and the method comprises: by existence with there is not candidate compound and deposit step acetylcholine receptor and the polypeptide described in any one of the present invention being carried out in case contacting; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.When α B-conotoxin VxXXIIIA can specific blockage α 9 α 10 acetylcholine receptor time, then infer that this acetylcholine receptor is the acetylcholine receptor of α 9 α 10 hypotype.

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

Another aspect of the invention relates to the purposes of polypeptide in the medicine preparing blockage of acetylcholine receptor or reagent described in any one of the present invention; Particularly, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.

Another aspect of the invention relates to polypeptide described in any one of the present invention medicine in preparation treatment or prevention nervous system disorders such as neurodynia, mammary cancer, lung cancer, habituation, parkinsonism or dementia etc., or the purposes of medicine for the preparation 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 wound, acquired immune deficiency syndrome (AIDS), head nerves is paralysed, drug intoxication, industrial pollution is poisoning, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic idiopathic esthesioneurosis, acute severe idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, chronic respiratory failure, composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film is scorching, allergy etc.

Another aspect of the invention relates to a kind of method treating and/or preventing nervous system disorders such as neurodynia, mammary cancer, lung cancer, habituation, parkinsonism or dementia etc., or kill pests, analgesia, smoking cessation or a method of quitting drug abuse, comprise the step of polypeptide of the present invention (conotoxin peptide or its propetide) or the pharmaceutical composition of the present invention giving significant quantity, particularly, described neurodynia is caused by following reason: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation wound, acquired immune deficiency syndrome (AIDS), head nerves is paralysed, drug intoxication, industrial pollution is poisoning, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic idiopathic esthesioneurosis, acute severe idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, chronic respiratory failure, composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, 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, by playing a role in conjunction with α 9 α 10 acetylcholine receptor (nAChR), has analgesic activities.Can be applicable to the nervous system disorders such as research, Diagnosis and Treat neurodynia, mammary cancer, lung cancer, parkinsonism, dementia, habituation and as useful molecular probe for aspects such as researchs.Different α class CTX is different to the affinity of vertebrate recipient, has the several order of magnitude of phase difference.Difference between this germline makes α class CTX can be used as useful probe for studying the germline generation of vertebrates nAChR, can be used as molecular probe to determine the different subtype of nAchR.They are the drug candidate of new drug development, lead drug and medicine.

Shown below is 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 is around or the pain that causes of former of central nervous system or secondary lesion or dysfunction or of short duration disorder, shows as spontaneous pain, allodynia, hyperpathia etc.A lot of disease all can cause neurodynia, comprise cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation wound, acquired immune deficiency syndrome (AIDS), head nerves is paralysed, drug intoxication, industrial pollution is poisoning, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic idiopathic esthesioneurosis, acute severe idiopathic neuralgia, extruding neurodynia, vasculitis (vasculitis)/local asphyxia, uremia, children's bile hepatic diseases, chronic respiratory failure, composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, 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 multiple regulating and controlling sequence comprising nucleotide sequence of the present invention and be operatively connected with it, described regulating and controlling sequence can instruct encoding sequence to express in suitable host cell under its consistency condition.Express be understood to include polypeptide produce in involved 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 their are separated from natural gene, or modified and containing combining and nucleic acid fragment arranged side by side in non-natural mode.When nucleic acid construct comprise express encoding sequence of the present invention required all regulating and controlling sequences time, term nucleic acid construct and expression cassette synonym.Term " encoding sequence " is defined as the part of the aminoacid sequence directly determining its protein product in nucleotide sequence in the text.The border of encoding sequence normally holds the ribosome bind site (for prokaryotic cell prokaryocyte) of opening code-reading frame upstream and next-door neighbour mRNA 3 ' to hold the transcription termination sequence in opening code-reading frame downstream to determine by next-door neighbour mRNA 5 '.Encoding sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Can the nucleotide sequence of the separation of operate coding peptide of the present invention in many ways, make it express described peptide.May expect or must process nucleotide sequence before insertion vector, this depends on expression vector.The technology of application 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, the termination signal that regulating and controlling sequence will comprise promotor and transcribe and translate.In order to import specific restriction site to be connected the coding region of regulating and controlling sequence with the nucleotide sequence of coded polypeptide, the regulating and controlling sequence of belt lacing can be provided.Term " is operatively connected " and is defined as so a kind of 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, with the expression making regulating and controlling sequence instruct 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 direct polypeptide is expressed.Promotor can be any nucleotide sequence having transcriptional activity in selected host cell, comprise sudden change, brachymemma with the promotor of heterozygosis, 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 can be obtained.

Regulating and controlling sequence can also be suitable transcription termination sequence, can be stopped one section of sequence of transcribing by host cell identification.Terminator sequence is operatively connected 3 ' end of the nucleotide sequence at coded polypeptide.Any terminator that can play function in selected host cell may be used to the present invention.

Regulating and controlling sequence can also be suitable leader sequence, namely very important to the translation of host cell mRNA non-translational region.Leader sequence is operatively connected 5 ' end of the nucleotide sequence in coded polypeptide.Any leader sequence that can 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 encode one section and be connected in the N-terminal aminoacid sequence of polypeptide in this district, coded polypeptide can be guided to enter emiocytosis approach.5 ' the end in nucleic acid sequence encoding district may natural containing translation frame as one man with the signal peptide coding region of the coding domain segment Nature Link of secrete polypeptide.Or it is external signal peptide coding region that 5 ' end of coding region can contain encoding sequence.When encoding sequence is not under normal circumstances containing signal peptide coding region, may need to add extraneous signal peptide-coding region.Or, natural signal peptide coding region can be replaced simply to strengthen polypeptide secretion with external signal peptide coding region.But any signal peptide coding region that can the polypeptide after expressing be guided to enter the Secretory Pathway of host cell used may be used to the present invention.

The all right Shi Tai original encoding district of regulating and controlling sequence, this district coding is positioned at one section of aminoacid sequence of amino terminus.Gained polypeptide is called as proenzyme or propolypeptide.Propolypeptide does not have activity usually, can be former and be converted into ripe active polypeptide from propolypeptide cutting peptide by catalysis or self-catalysis.

The N-terminal of polypeptide not only have signal peptide but also You Taiyuan district time, the N-terminal of Tai Yuan district next-door neighbour polypeptide, signal peptide district is then close to the N-terminal in Tai Yuan district.

Interpolation can regulate the regulating and controlling sequence of expression of polypeptides to be also needs according to the growing state of host cell.The example of regulator control system is that those can be reacted to chemistry or physical stimulation thing (when being included in regulating compound), thus system that is open or closedown genetic expression.Other examples of regulating and controlling sequence are that those can make the regulating and controlling sequence of gene amplification.In these examples, together with the nucleotide sequence of coded polypeptide should being operatively connected with regulating and controlling sequence.

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 and prepare recombinant expression vector, this carrier can comprise 1 or multiple restriction site easily, to insert or to replace the nucleotide sequence of coded polypeptide in these sites.Or, nucleotide sequence of the present invention can be expressed by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted suitable expression vector.When preparing expression vector, encoding sequence can be made to be arranged in carrier to be operatively connected with suitable expression regulation sequence.

Recombinant expression vector can be that any being convenient to carries out recombinant DNA operation and the carrier (such as plasmid or virus) of express nucleic acid sequence.Carrier and its consistency of host cell that will import are depended in the selection of carrier usually.Carrier can be linear or closed circular form plasmid.

Carrier can be self-replicating type carrier (is namely present in extrachromosomal complete structure, can copies independent of karyomit(e)), such as plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can comprise any mechanism ensureing self-replacation.Or, carrier be one when importing host cell, will to be incorporated in genome and the carrier copied together be incorporated into karyomit(e).In addition, single carrier or plasmid can be applied, or totally comprise two or more carrier or the plasmid of all DNA by importing host cell gene group, or transposon.

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

Preferred carrier of the present invention comprises can make carrier stabilizes be incorporated in host cell gene group, or ensures that carrier carries out the element of self-replicating in cell independent of cellular genome.

With regard to carrying out the situation of self-replicating, carrier can also comprise replication orgin, and carrier independently can be copied in target host cell.Replication orgin can with the sudden change making it become responsive to temperature type in host cell (see such as, fEhrlich, 1978, NAS journal 75:1433).

More than 1 nucleotide sequence of the present invention copied can be inserted to improve the output of this gene product to host cell.The copy number increase of this nucleotide sequence can be passed through at least 1 of this sequence additional copies Insertion Into Host Cell genome, or a selective marker that can increase is inserted together with this nucleotide sequence, by culturing cell under having appropriate selectable agent to exist, pick out the selected marker containing amplification copy thus the cell containing additional copies nucleotide sequence.

For connect above-mentioned each element to build the operation of recombinant expression vector of the present invention be well-known to those skilled in the art (see such as Sambrook etc., Molecular Cloning: A Laboratory handbook, the second edition, CSH Press, cold spring port, New York, 1989).

Host cell

The invention still further relates to the recombinant host cell comprising the nucleotide sequence of the present invention that can be used to recombinant production polypeptide.Can the vector introduction host cell of the nucleotide sequence of the present invention be comprised, thus this carrier is maintained with the outer carrier format of karyomit(e) of above-mentioned chromosomal integrant or self-replacation.Any offspring different from parental cell due to the sudden change occurred between replicative phase contained in term " host cell ".Polypeptide coding genes 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, such as bacterium or yeast cell.Can by technology well known to those skilled in the art by vector introduction host cell.

Preparation method

The invention still further relates to the method that peptide of the present invention is prepared in restructuring, the method comprises: (a) is being suitable under the condition producing described peptide, and cultivate the host cell containing nucleic acid construct, this nucleic acid construct comprises the nucleotide sequence of coding for said peptides; (b) this peptide is reclaimed.

In preparation method of the present invention, culturing cell in the nutritional medium produced at appropriate polypeptides by means known in the art.Such as; can in suitable substratum; under the condition allowing expression of polypeptides and/or separation, carry out 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 substratum comprising carbon and nitrogenous source and inorganic salt, step known in the art is adopted to cultivate.Suitable substratum can be provided by supplier or the catalogue of American type culture collection (such as, described in) can prepare with reference to disclosedly forming.If polypeptide is secreted in substratum, then directly can reclaim polypeptide from substratum.If polypeptide is not secreted, can reclaim from cell lysate.

The polypeptide produced can be reclaimed by means known in the art.Such as, routine operation (including, but are not limited to centrifugal, filtration, extracting, spraying dry, evaporation or precipitation) can be passed through from substratum, reclaim polypeptide.

Purifying polypeptide of the present invention can be carried out by various operation known in the art, these operations comprise, but be not limited to chromatography (such as, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography), HPLC, electrophoresis (such as, the isoelectric focusing of preparation property), differential solubility (such as ammonium sulfate precipitation), SDS-PAGE or extracting be (see such as, protein purification, J.C.Janson and Lars Ryden compiles, VCH Publishers, New York, 1989).

Transgenic animals and plants

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

For the method and formulation of Control pests

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

Can by preparation containing conotoxin peptide or comprise polynucleotide of the present invention the formulation application of recombinant microorganism in soil.The product of preparation can also be covered the whole plant process application in material or root process or crop cycle late period as seed.Preparation can comprise spreader-sticker adjuvants, stablizer, other insecticidal additive or tensio-active agent.Liquid preparation can be based on water or non-water, and uses 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 the person's character due to special preparation extensively changes by insecticide concentration, 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 the normally about 1-60% of solid weight in liquid phase.Preparation containing cell will usually containing about 10 ²-about 10 ⁴individual cell/mg.These preparations will use with the amount of the about 50mg of per hectare (liquid or dry)-1kg or more.By spraying, spreading, spill, etc., can by formulation application such as, in insect environment, soil and plant.

Pharmaceutical composition

The invention still further relates to the pharmaceutical composition containing peptide of the present invention and pharmaceutical acceptable carrier and/or vehicle.Described pharmaceutical composition can be used for research, diagnosis, alleviates or treats the disease relevant with neurodynia, mammary cancer, lung cancer, amentia, habituation, pain, parkinsonism, psychosis, depression, myasthenia gravis etc. or illness.In one embodiment, the pharmaceutical composition of peptide of the present invention containing treatment significant quantity is beneficial to medicinal mode and prepares and administration, and need consider individual patient clinical condition, transport the known other factors of site, medication, administration schedule and doctor.Therefore " significant quantity " for this paper object is determined by the consideration of these aspects.

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

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

Can apply conotoxin peptide of the present invention as useful probe for zoologize nAChR germline occur; The different subtype of nAChR is determined as molecular probe; As molecular model, design new drug; As research, diagnosis nervous system disease as the instrument medicine of Parkinson's disease, action obstacle, schizophrenia etc. and medicine; The candidate agent for the treatment of mammary cancer, lung cancer, small cell lung cancer etc.As polypeptide pesticide, be developed as novel biopesticide etc.

The beneficial effect of the invention

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

Accompanying drawing explanation

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

Fig. 2: display be α B-VxXXIIIA mature peptide sequence (SEQ ID NO:1) and the 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.

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

Fig. 4: display be 10 μMs of α B-VxXXIIIA12 to α 9 α 10, α 7, α 3 β 4, affect situation with the electric current of α 4 β 2nAChRs.The contrast electric current that in figure, " C " refers to, immediately " C " below be the current locus that α B-VxXXIIIA12 blocks first Ach pulse formation of corresponding receptor subtype.10 μMs of α B-VxXXIIIA12 specific blockage α 9 α 10nAChR, and do not block α 7 completely, α 3 β 4, α 4 β 2nAChRs hypotype.

Fig. 5: display be with barium ion ND96 perfusate (Ba ⁺⁺-ND96) when replacing conventional ND96 perfusate, α B-VxXXIIIA12 is to the concentration dose response curve of α 9 α 10nAChR, and in figure, X-coordinate is the logarithmic value (Log [Toxin Concentration] M) of the volumetric molar concentration (M) of 3 α B-VxXXI I IA isomer used; Ordinate zou is dose response percentage ratio (%Response), is acetylcholine receptor electric current and the ratio percentage ratio contrasting electric current under the detoxifying function of respective concentration.The blocking-up activity of VxXXIIIA12 to α 9 α 10nAChR is the strongest.With etc. the barium ion ND96 perfusate of volumetric molar concentration replace in conventional ND96 perfusate calcium ion (Ca ⁺⁺), Xenopus Oocytes effectively can be stoped to be activated by flow of calcium ions and to produce chlorion (Cl ^-) 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 for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press), corresponding reference or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

embodiment 1: the extraction of cone shell poison pipe total serum IgE and the structure of cDNA library thereof

From Hainan Island, the Xisha Islands is coastal collects calamus cone shell (Conus vexillum); extract the total serum IgE of calamus cone shell poison pipe with a small amount of post centrifugal tissue/cell total serum IgE extraction agent box (Shanghai Hua Shun biotechnology company limited), operate according to test kit specification sheets.By the total serum IgE extracted, scan under 200 ~ 300nm wavelength with PE company nucleic acid-protein analyser, detect content and the purity of RNA; The integrity of RNA is detected with 1.1% agarose gel electrophoresis.

By Clontech company Creator ^tMsMART ^tMcDNA library builds test kit specification sheets and builds calamus cone shell poison pipe cDNA library, is summarized as follows: get 3 μ L (about 1.0 μ g) total serum IgE, adds CDSIII/3 ' PCR Primer (1 μ L), SMARTIV ^tMoligonucleotide (1 μ L), 72 DEG C of incubation 2min.Add 5 × First-Strand Buffer (2.0 μ L) again, 1.0 μ L dNTPMix (10mmol/L), 1 μ L DTT (20mmol/L), PowerScript ThermoScript II (1 μ L).42 DEG C of temperature bath 1h synthesize cDNA first chain.With cDNA first chain for template, with CDSIII/3 ' PCR primer and 5 ' PCR for primer, with LD-PCR (long-distance PCR) synthetic double chain cDNA.Reaction conditions: 95 DEG C of 20s, 95 DEG C of 5s, 68 DEG C of 6min, the agarose electrophoresis with 1.1% detects the quality of double-strand cDNA synthesis.After PCR primer purifying, cut through SfiI enzyme, CHROMA SPIN-400 post carries out fractional separation; After separation, often 3 μ L detect cDNA size distribution with 1.1% agarose gel electrophoresis got by pipe, and collection tube cDNA fragment being greater than 500bp merges, resuspended for subsequent use with 7 μ L deionized waters after purifying.

Get 1.5 μ L double-strand cDNA and 1.0 μ L pDNR-LIB (0.1 μ g/mL, the carrier of SfiI process), 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 DEG C of connections are spent the night.After connecting product purification, with electroporation, recombinant plasmid is imported competent cell intestinal bacteria JM-109 ^[8].Collect satisfactory transformation mixture, thus form original cDNA library, after amplification, obtain satisfactory cDNA library.

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

(Kaas, Q. that 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 conotoxin of same superfamily, and there is identical halfcystine pattern.This conservative property is that the discovery of a certain specific superfamily conotoxin newcomer and qualification provide effective way (Zhangsun, D.; 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 & drug design 2006,68 (5), 256-65.).

The mono-clonal of the present invention's random picking about 50 from calamus cone shell poison pipe cDNA library carries out sequencing analysis, institute's cDNA sequence that obtains, through 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 carry out analyzing (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites.Protein engineering, design & selection:PEDS 2004,17 (1), 107-12.).We have therefrom found a unique cDNA sequence (SEQ ID NO:3), containing 1 open reading frame (SEQ ID NO:4), the propeptide sequence (SEQ ID NO:2) that its coding produces is only containing signal peptide and mature peptide (SEQ ID NO:1) two integral parts, this and all conotoxin precursor peptides in the past found are by signal peptide, propetide, mature peptide 3 integral parts are all different, there are 4 unique halfcystine patterns " C-CC-C ", these are all different from the halfcystine pattern of all conotoxins found in the past, signal peptide sequence is all different from the signal peptide of known superfamily, what thus this SEQ ID NO:3cDNA encoded is a new superfamily conotoxin, our called after B-superfamily, 23rd (XXIII) plants halfcystine pattern, and be the conotoxin of first this superfamily identified from calamus cone shell.

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

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

Precursor peptide aminoacid sequence shown in SEQ ID NO:2 (hereinafter 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 SEQ ID NO:3: GACTCCTATTGTCATA atgGAGACTCTCACTCTACTATGGCGAGCGTCGTCGTCTTG cCTGCTCGTCGTTCTCAGTCACTCGCTTCTTCGTCTTCTCGGGGTTAGGTGTTTGG AGAAG aGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTGTCAAAAGGGCCCTTC TTTCG cCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAGtaatCCGGGGGGTCCGAAATGTGAAATAAGAAGGCCGAGCATACAACCCTCCTTAACAC TCGCCTATTGTAAAAGGGTGTCCTTTGTCAATAGTGGTACTAATGTTGTTTAAAAA ATCCGCTGTGATATCTTAGGTGTTCAAGTAGTAGGTTGCACCCCCCTCGAGAGGGT ATACCAGCTGGTCGTCCGCCGGCTATTAAGTGATCACTCACCCTTTGTTGATGGTG GTACAAGTTAAAGGATCCCCCGGGCGCGTGGTTTTCTTACCACATTTTTAGAAGTC CCCCCGGCGCCGCCGGTTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

Open reading frame (ORF) polynucleotide sequence of the coding VxXXIIIA precursor protein shown in SEQ ID NO:4: atgGAGACTCTCACTCTACTATGGCGAGCGTC gTCGTCTTGCCTGCTCGTCGTTCTCAGTCACTCGCTTCTTCGTCTTCTCGGGGTTA GGTGT tTGGAGAAGAGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTGTCAAAA GGGCC cTTCTTTCGCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAGtaa.

The polynucleotide sequence of the coding VxXXIIIA mature peptide shown in SEQ ID NO:5: gTT aGGTGTTTGGAGAAGAGCGGCGCGCAACCTAATAAGTTATTTCGTCCGCCGTGCTG TCAAA aGGGCCCTTCTTTCGCCCGTCACTCGCGTTGCGTTTATTACACTCAATCGAGGGAG taa.

the synthetic of embodiment 3: α B-conotoxin VxXXIIIA

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

The resin peptide of three isomer adopts Fmoc chemical process to carry out synthetic, except halfcystine, and the side chain protected group of all the other amino acid standards.-SH Trt (S-trityl) protection of the 1st and the 2nd halfcystine (Cys) of VxXXIIIA12 ,-the SH of the 3rd and the 4th halfcystine protects in pairs with Acm (S-acetamidomethyl);-SH Tr t (S-trityl) protection of the 1st and the 3rd halfcystine (Cys) of VxXXIIIA13 ,-the SH of the 2nd and the 4th halfcystine protects in pairs with Acm (S-acetamidomethyl);-SH Trt (S-trityl) protection of the 2nd and the 3rd halfcystine (Cys) of VxXXIIIA14 ,-the SH of the 1st and the 4th halfcystine protects in pairs with Acm (S-acetamidomethyl).Its synthesis step is: adopt Fmoc and the FastMoc method in solid-phase synthesis, ABI Prism 433a Peptide synthesizer has synthesized 3 isomer linear peptides.The amino acid whose Side chain protective group of Fmoc is: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr); OBut (Asp); Boc (Lys). adopt Fmoc HOBT DCC method; Rink amidated resin and Fmoc amino acid, synthesis step reference instrument synthesis handbook carries out.For reacting completely, difference proper extension on piperidines deprotection and Coupling time, connects amino acid to difficulty and adopts two coupling, 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 ice ether sedimentation and washing and recycling linear peptides crude product, with preparative reverse hplc C18 post (Vydac) purifying, wash-out linear gradient is that 20-60%B60. solvent B is 60%CAN (acetonitrile) in 0-40min, 40%H20,0.92%TFA (trifluoroacetic acid); Solvent orange 2 A is the aqueous solution of 1%TFA.

The HPLC C18 post (Vydac) of the linear peptides analysis mode after purifying carries out purity detecting, and gradient is 0-50min 0-50%buffer B, 50-55min 50-100%bufferB, and flow velocity is 1mL/min.Its purity reaches more than 95%, 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.) carry out the folding reaction of two-step oxidation to the linear peptides of 3 isomer VxXXIIIA12, VxXXIIIA13 and VxXXIIIA14, process is summarized as follows:

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

The determining molecular weight that the theoretical molecular (monoisotopic mass) of 3 isomer after oxidative folding is 4622.27Da, VxXXIIIA12 is 4622.3Da; The determining molecular weight of VxXXIIIA13 is 4622.2Da; The determining molecular weight of VxXXIIIA14 is 4622.4Da.Peptide concentration colorimetric estimation under 280nm wavelength, calculates peptide concentration and quality 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 tests

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): 11625-32.) method in, and in-vitro transcription test kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) specification sheets, prepare various rat 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), the OD value under its concentration UV 260nm is calculated.Dissect and collect Africa xenopus (Xenopus laveis) ovocyte (frog's egg), be injected into by cRNA in frog's egg, the injection volume of each subunit is 5ng cRNA.Muscle nAChR each subunit injection 0.5-2.5ng DNA.Frog's egg is cultivated in ND-96.Injection cRNA in 1-2 days after frog's egg collection, the 1-4 days interior voltage-clamp recording for nAChRs after injection.

The frog's egg 1 being injected cRNA is placed in the Sylgard track of 30uL (diameter 4mm × degree of depth 2mm), 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 containing 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) free switching can be carried out between perfusion toxin or vagusstoff (ACh), and a series of threeway solenoid valve (solenoid valves, model161TO31, Neptune Research, Northboro, MA) make to carry out free switching between perfusion ND96 and ACh etc.The electric current of Ach gate is by two-electrode voltage pincers amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) be arranged on " slowly " pincers, 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 voltage and current electrode.Membrane voltage strangulation in-70mV. whole system by conputer controlled and record data.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 μMs; The α 7 expressing the nAChRs of nervous system type is 200 μMs, and other hypotype is all 100 μMs.At least record 4 ovum and express the current response situation of certain hypotype to different toxin concentration, and current locus.

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

Result shows, 10 μMs of α B-VxXXIIIA12 (prepared by embodiment 3) have almost blocked completely by the open electric current produced of 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).Their half block dosage IC50 and limit of error is respectively α B-VxXXIIIA12,1.2 μMs (0.8-1.7 μM); α B-VxXXIIIA13,3.9 μMs (2.7-5.6 μM); α B-VxXXIIIA14 > 30 μMs. the slope (Hillslope) of their dose response curve and limit of error are respectively VxXXIIIA12,1.4 (0.5-2.1) and VxXXIIIA13,1.3 (0.9-1.7). therefore, the follow-up blocking-up for other nAChRs hypotypes is active, just only detects with the strongest active α B-VxXXIIIA12 isomer.The slope that partly block dosage IC50 and dose response curve of α B-VxXXIIIA12 to various nAChRs hypotype is as shown in table 1.

Table 1: α B-VxXXIIIA12 blocks the slope of dosage IC50 and dose response curve to half of various nAChRs hypotype

Note: a to be degree of confidence be 95% interval.

The blocking-up selectivity of α B-VxXXIIIA12 to α 9 α 10nAChR is high.From 10 μMs of α B-VxXXIIIA12 to α 9 α 10, α 7, α 3 β 4, affect situation with the electric current of α 4 β 2nAChRs can find out (Fig. 4), α B-VxXXIIIA12 specific blockage α 9 α 10nAChR, and the toxin of same concentrations is to α 7, α 3 β 4, with α 4 β 2nAChRs hypotype without any blocking activity.

Known α 9 α 10nAChR is to calcium ion (Ca ⁺⁺) there is very high permeability.Can be activated by the flow of calcium ions of nAChRs and produce chlorion (Cl ^-) outflow electric current, on Xenopus Oocytes, this electric current accounts for more than 90% of the open electric current of the α 9 α 10nAChR observed.On the contrary, close with calcium ion barium ion (Ba ⁺⁺) generation Cl-currents can not be activated.Thus, 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 observed 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).Their half 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 (Hillslope) of their dose response curve 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 to similar containing the result under the normal ND96 perfusate condition of calcium ion.Therefore, VxXXIIIA has blocked α 9 α 10nAChR really, instead of blocks because of calcium ion activated Cl-currents.

Research shows, α 9 α 10nAChR is novel targets (McIntosh, the 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 & 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, α B-of the present invention new superfamily conotoxin VxXXIIIA has high using value in the study mechanism of above-mentioned disease, diagnosis, 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 amendment 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 (19)

1. a peptide species, its aminoacid sequence is the aminoacid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

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

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

4. polynucleotide according to claim 3, its nucleotides sequence is classified as the nucleotide sequence be selected from according to any one of following (1) to (2):

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

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

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

6. an expression vector, it comprises nucleic acid construct according to claim 5.

7. the cell transformed, it comprises expression vector according to claim 6.

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

9. a pharmaceutical composition, it comprises the polypeptide described in claim 1 or 2, or comprises fusion rotein according to claim 8.

10. pharmaceutical composition according to claim 9, it also comprises pharmaceutically acceptable carrier or auxiliary material.

11. 1 kinds of blockage of acetylcholine receptor or regulate the method for levels of acetylcholine in vitro, comprise the step of the polypeptide described in claim 1 or 2 using significant quantity.

12. methods according to claim 11, wherein, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.

13. 1 kinds of methods of screening acetylcholine receptor inhibitor or determining acetylcholine receptor subtypes, the method comprises: by existence with there is not candidate compound and deposit step acetylcholine receptor and the polypeptide described in claim 1 or 2 being carried out in case contacting.

14. methods according to claim 13, wherein, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.

The purposes of polypeptide described in 15. claims 1 or 2 in the medicine preparing blockage of acetylcholine receptor or reagent.

16. purposes according to claim 15, wherein, described acetylcholine receptor is α 9 α 10 acetylcholine receptor.

Polypeptide described in 17. claims 1 or 2 treats and/or prevents the purposes of medicine of neurodynia, mammary cancer, lung cancer or wound healing in preparation, or for the preparation of the purposes of analgesic.

18. purposes according to claim 17, wherein, described neurodynia is caused by following reason: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, zoster, mechanical injury and operation wound, acquired immune deficiency syndrome (AIDS), head nerves is paralysed, drug intoxication, industrial pollution is poisoning, lymph neurodynia, myelomatosis, multiple spot motorius pain, chronic idiopathic esthesioneurosis, acute severe idiopathic neuralgia, extruding neurodynia, vasculitis, vasculitis, local asphyxia, uremia, children's bile hepatic diseases, chronic respiratory failure, composite nerve pain, multiple organ failure, sepsis/pyemia, hepatitis, porphyria, vitamin deficiency, Chronic Liver popular name for, primary bile sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory nerve bundle film inflammation or allergy.

The preparation method of the polypeptide described in 19. claims 1 or 2, 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) or Acm (Cys), But (Thr), But (Ser), But (Tyr), OBut (Asp), Boc (Lys); Between corresponding halfcystine, fixed point forms disulfide linkage respectively;

2) by 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 preparative reverse hplc C18 column purification;

3) by step 2) in the product that obtains carry out two-step oxidation and fold.