WO2014023129A1 - α-芋螺毒素肽、其药物组合物及用途 - Google Patents

α-芋螺毒素肽、其药物组合物及用途 Download PDF

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WO2014023129A1
WO2014023129A1 PCT/CN2013/077363 CN2013077363W WO2014023129A1 WO 2014023129 A1 WO2014023129 A1 WO 2014023129A1 CN 2013077363 W CN2013077363 W CN 2013077363W WO 2014023129 A1 WO2014023129 A1 WO 2014023129A1
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polypeptide
seq
acetylcholine receptor
neuralgia
nachrs
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PCT/CN2013/077363
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French (fr)
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罗素兰
长孙东亭
吴勇
朱晓鹏
胡远艳
邴晖
麦金托什•J.•迈克尔
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海南大学
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Priority claimed from CN201210277619.8A external-priority patent/CN103570808B/zh
Priority claimed from CN201210325531.9A external-priority patent/CN103665130B/zh
Priority claimed from CN201210347966.3A external-priority patent/CN103665133B/zh
Application filed by 海南大学 filed Critical 海南大学
Priority to EP13828357.7A priority Critical patent/EP2889307B1/en
Priority to JP2015525711A priority patent/JP6336979B2/ja
Priority to US14/419,584 priority patent/US9469674B2/en
Publication of WO2014023129A1 publication Critical patent/WO2014023129A1/zh

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    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/944—Acetylcholine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention is in the field of biochemistry and molecular biology and relates to a novel ⁇ -conotoxin peptide, a pharmaceutical composition thereof, a preparation method thereof and use thereof.
  • the present invention also relates to a propeptide of the conotoxin peptide, a nucleic acid construct thereof, an expression vector thereof, and a transformed cell, and a fusion protein thereof.
  • the invention also relates to a method of blocking an acetylcholine receptor, and to a pharmaceutical use of the spirulina peptide. Background technique
  • Conotoxin (Conotoxin, Conopeptide, CTx) secreted by the carnivorous mollusk, Comis, which lives in the tropical ocean, has special functions for regulating various ion channels and has been shown to be important in clinical practice. value.
  • Conotoxin usually contains 10 - 46 amino acids, is rich in disulfide bonds, has strong biological activity, and can specifically act on receptors and ion channels on animal cell membranes. In particular, it has high selectivity for voltage-gated or ligand-gated ion channels (including a few G-protein-related receptors, etc.).
  • Conotoxin is divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence of its precursor protein and the cysteine pattern.
  • all known conotoxins can be divided into 19 superfamilies.
  • Conotoxin can be divided into various pharmacological families such as ou ⁇ , ⁇ , ⁇ according to its receptor target. Each superfamily can be divided into ⁇ , ⁇ , ⁇ ( ⁇ -superfamily) according to the type of receptor target, ⁇ , ⁇ , ⁇ :, ⁇ ( ⁇ -superfamily), ⁇ , ⁇ , KM (M-super Family) (family). Among them, ⁇ -conotoxin is the most selective selective nicotinic acetylcholine receptor (nAChRs) subtype specific blocker.
  • nAChRs nicotinic acetylcholine receptor
  • ⁇ -conotoxin and its target nAChRs are of great value in the study of various disease mechanisms and drug development.
  • ⁇ -conotoxin is one of the earliest discovered conotoxins, usually with a small molecular mass, generally consisting of 12
  • A-conotoxin has a wide variety of activities, diverse activities, and complex structural changes. A-conotoxin can be classified by its highly conserved signal peptide sequence, pharmacological activity and cysteine pattern.
  • the cysteine mode of a-conotoxin is CC-C-C, in which the disulfide bond of the natural peptide is C1-C3 and C2-C4, which is called the spherical isomer.
  • Spherical isomers have complete biological activity, and banded isomers sometimes exert biological activity through different mechanisms of action, and the activity of bead-like isomers tends to decrease.
  • Two loop loops are formed between the disulfide bonds, and the ⁇ -conotoxin can be divided into ⁇ 3/5, «4/7, «4/6, according to the different amounts of the two-three and three-tetracysteine-based amino acids.
  • a variety of subfamilies such as ⁇ 4/4 and ⁇ 4/3, the difference in the characteristics and residue composition of each loop is that the toxin acts on different receptor subtypes ⁇ fili ( Ulens C, Hogg RC, Celie PH, et al.
  • Nicotinic acetylcholine receptors are cell membrane proteins with important physiological and clinical significance in the animal kingdom. They are the first type of receptors discovered in humans and can be divided into two types: muscle acetylcholine receptors and neurotypes. Acetylcholine receptor. nAChRs are allosteric membrane proteins on the cell membrane that mediate the physiological functions of many central and peripheral nervous systems, including learning, memory, addiction, response, analgesia, and motor control. nAChRs activate dry release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-acetylbutyric acid.
  • nAChRs have been shown to be key targets for screening and treating a wide range of important disease drugs, including pain, alcohol and tobacco addiction, mental retardation, dementia, schizophrenia, central nervous disorders, epilepsy, Parkinson's disease, Mental illness, neuromuscular block, myasthenia gravis, depression, high blood pressure, arrhythmia, asthma, muscle relaxation, stroke, breast cancer and lung cancer.
  • drugs for symptomatic treatment of the above diseases There are no drugs for symptomatic treatment of the above diseases.
  • Commonly used non-selective nAChRs agonists such as nicotine, while alleviating the symptoms of these neurological diseases, have strong side effects on the heart and gastrointestinal tract and are addictive.
  • the premise of developing such a drug is to obtain a selective compound that can specifically bind to various subtypes of nAChRs, as a tool to study and identify the fine composition and physiological functions of various subtypes, or directly as a related disease. medicine.
  • Activation promotes tumor cell proliferation, and the activation of these receptors by drugs can be used for early diagnosis or treatment of these catastrophic cancers.
  • nAChRs are assembled from different alpha and beta subunits into f subtypes, each with distinct pharmacological characteristics.
  • the muscle acetylcholine receptor consists of 5 subunits, containing 2 ⁇ subunits, 1 ⁇ subunit, 1 ⁇ subunit and 1 ⁇ or ⁇ subunit. The ⁇ or ⁇ subunit depends on whether it is Fetal or adult acetylcholine receptor.
  • the mammalian neuronal nAChRs subtype is much more complex than the muscular nAChRs, with at least 8 alpha subunits and 3 beta subunits, al-a, ⁇ 9, ⁇ ( ⁇ 8 in chicks), and ⁇ 2- 44.
  • «2, «3 and ⁇ 4 can bind to ⁇ 2 or ⁇ 4, respectively, to form functional receptors, such as ⁇ 2 ⁇ 2, ⁇ 3 ⁇ 2, ⁇ 2 ⁇ 4, etc.; ⁇ 9 and ⁇ combine to form functional receptor ⁇ 9 ⁇ 10 nAChRs. In addition, ⁇ 7 and «9 can form homomultimers. Due to the lack of highly selective ligand compounds for various subtypes, there are many challenges to study and elucidate the fine structure and function of the various nAChRs subtypes.
  • Drug addiction is both a medical problem and a serious social problem.
  • nAChRs nicotinic acetylcholine receptor
  • niChRs of dopaminergic (DA) neurons in the table i1 ⁇ 2 It is a drug target for the treatment of neuropsychiatric diseases such as nicotine, morphine and cocaine addiction, Parkinson's disease, dementia, schizophrenia, depression, etc.
  • DA dopaminergic
  • Egecioglu E.; Dickson, SL; Svensson, L.; Engel, JA, Alpha-conotoxin Mil-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus accumbens. European neuropsychopharmacology, 2008, 18 (7), 508-18).
  • ⁇ -conotoxin Mil which blocks ⁇ 3 ⁇ 2 and ⁇ 6 ⁇ 2* nAChRs, partially and differentially blocks nicotine-induced dopamine release in the striatum.
  • Presynaptic nAChRs contain at least two subtypes, namely Mil-sensitive Type and Mil-insensitive, DA dry release of dopaminergic neurons (Kaiser SA, Soliakov L, Harvey SC, Luetje CW, Wonnacott S. Differential inhibition by ⁇ -conotoxin-MII of the nicotinic stimulation of [3H] dopamine release From rat striatal synaptosomes and slices. J Neurochem 1998; 70: 1069-76 ).
  • nAChRs containing ⁇ 6 subunits in DA neurons are very high.
  • the mechanism of the important role of ⁇ 6 nAChR in addiction is unclear due to the lack of pharmacological molecular probes specific for ⁇ 6* nAChRs.
  • the 3 ⁇ 46p2*-nAChRs subtype in the striatum of mammalian brain is considered to be a drug target for the treatment of cravings and drug addiction (Exley, R.; Clements, MA; Hartung, ⁇ ; Mcintosh, J.
  • A6* nAChRs also express i1 ⁇ 2 catechins on the amine and the retina (Le Novere, N.; Zoli, ⁇ ; Changeux, JP, Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain.
  • ⁇ 6 ⁇ 2* nAChRs exhibits a function of regulating dopamine release, in primate The number of ⁇ 6 ⁇ 2* nAChRs in the 1-methyl-4-phenyl- 1 , 2, 3, 6-tetr ahydropyridine animal model and the human Parkinson's disease model was significantly reduced (Champtiaux, N.; Han, Z.
  • a specific blocker of 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs is a valuable tool for studying the physiological functions of ⁇ 6* nAChRs present in different tissues, and a therapeutic drug or drug screening tool for diseases such as addiction and Parkinson's disease. medicine.
  • the pain affects about 1/6 of the population, including arthritis, neuralgia, swelling and pain.
  • neuropathic pain affects 4-8 % of people, including alcoholism, sciatica, cancer and cancer chemotherapy, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injuries and surgical injuries.
  • nAChRs containing (3 ⁇ 43-subunits, including ⁇ 3 ⁇ 2 and ⁇ 3 ⁇ 4 subtypes are mainly expressed in the peripheral nervous system, also distributed in the central nervous system, and are targets of neuropathic drugs.
  • ⁇ -conotoxin blocking ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 nAChRs Preclinical models of chronic pain have shown good analgesic activity and are not addictive. Intractable pain is a H3 ⁇ 4: boundary health problem, and new therapeutic drugs are urgently needed (apier, IA Klimis, ⁇ ; Rycroft, ⁇ . ⁇ ; Jin, ⁇ . ⁇ ; Alewood, PF; Motin, L.; Adams, DJ; Christie, MJ, Intrathecal ⁇ -conotoxins Vcl.l, AuIB and Mil acting on Neuropharmacology 2012,62 (7), 2202-2207. Blyth, F. ⁇ ; March, L.
  • 33 ⁇ 4 nAChRs are the major acetylcholine receptor subtypes in the sensory and autonomous neural centers. 33 ⁇ 4 nAChRs are also branches of central nervous system (CNS) neurons, such as the centrally extending pineal gland and dorsal bone marrow, which are involved in the addiction of nicotine and other drugs of abuse (Millar, NS; Gotti, C" Diversity of vertebrate Nicotinic acetylcholine receptors. Neuropharmacology 2009,56 (1), 237-46; Tapper, AR; McKinney, SL; Nashmi, R.; Schwarz, J.; Deshpande, P.; Labarca, C; Whiteaker, P.; Marks, MJ; Collins, A.
  • CNS central nervous system
  • ⁇ 3 ⁇ 4 nAChRs also plays an important role in fear response.
  • the role of regulation of glutamate and norepinephrine release (Zhu, PJ; Stewart, RR; Mcintosh, JM; Weight, FF, Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral Amygdala neurons. Journal of neurophysiology 2005,94 (5), 3081-91. Alkondon, ⁇ ; Albuquerque, EX, A non-alpha7 nicotinic acetylcholine receptor modulates excitatory input to hippocampal CAl interneurons.
  • ⁇ -CTxs which have extraordinary selectivity for specific subtypes of nAChRs, are absolutely essential tools for the study of the distribution and function of various subtypes and therapeutic agents for related diseases (Kasheverov, IE, Utkin, Y. ⁇ ., and Tsetlin, VI ( 2009) Naturally Occurring and Synthetic Peptides Acting on Nicotinic Acetylcholine Receptors. Current Pharmaceutical Design 15, 2430-2452; Nicke, A., Wonnacott, S., and Lewis, RJ (2004) alpha-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. Eur. J. Biochem.
  • nAChRs tends to overlap with the surface of ⁇ 6 ⁇ 2* nAChRs, and sometimes ⁇ 3 ⁇ 2* nAChRs are dominant (Whiteaker, P., Mcintosh, J. ⁇ , Luo, SQ, Collins, A. C” and Marks, MJ ( 2000) I-125-alpha-conotoxin Mil identifies a novel nicotinic acetylcholine receptor population in mouse brain. Molecular pharmacology 57, 913-925; Whiteaker, P., Peterson, CG, Xu, W., Mcintosh, JM, Paylor, R .
  • ⁇ 3 ⁇ 2* nAChRs in the spine plays an important role in the transmission of pain stimuli and is a target for analgesic drugs (Young, ⁇ , Wittenau Er, S., Mcintosh, JM, and Vincler, M. (2008) Spinal ⁇ 3 ⁇ 2* nicotinic acetylcholine receptors tonically inhibited the transmission of nociceptive mechanical stimuli. Brain research 1229, 118-124 ) A true ⁇ 3 ⁇ 2* vs. ⁇ 6 ⁇ 2* nAChRs The discovery of selective blockers is of great value for comprehensive research to understand the function and significance of this subtype under normal and disease states.
  • ⁇ -conotoxin has great potential in the fields of pain, smoking cessation, detoxification, treatment of new drugs such as Parkinson's disease, dementia, depression and schizophrenia, and research on related disease mechanisms.
  • the market is also in urgent need of distinguishing specific nAChRs.
  • One aspect of the invention relates to a polypeptide having the following amino acid sequence of formula I:
  • X 2 represents P, A or V
  • X 3 represents R, N or S
  • X 4 represents N, V or A
  • X 5 represents K, D, M or A
  • X 6 means H or S
  • X 8 represents L or I
  • X 9 indicates that G or X 9 is missing
  • the C-terminus of the polypeptide of Formula I is amidated.
  • the C-terminal amidation of a polypeptide of formula I can also be represented by #, ie:
  • the present invention also relates to a polypeptide which is or comprises an amino acid sequence selected from any one of (1) to (3) below: (1) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11-15, SEQ ID NO: 26-28 or SEQ ID NO: 30 Site acid sequence;
  • amino acid sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 97% identical to the above-mentioned (1) amino acid sequence;
  • the degree of identity between two or more amino acid sequences is determined by the BLAST 2.0 protein database query program (Aaltschul et al., 1997, Nucleic Acids Res. 25: 3389-3402) and determined using the following parameters.
  • the acid sequence may differ by substitution, insertion, addition and/or deletion of one or more, preferably from 1 to 5, more preferably from 1 to 3, particularly preferably from 1 to 2, most preferably 1 atmospheric acid residue base.
  • the amino acid change is a minor change in properties, i.e., a conservative acid substitution that does not significantly affect the folding and/or activity of the protein; small fragment deletions, usually from 1 to about 5, preferably 1 - a deletion of 3, more preferably 1 amino acid; a small aryl or carboxy terminal extension, such as a methionine residue added at the base; a small linker peptide of up to about 20-25 residues; Small extensions such as polyhistidine fragments, epitopes or binding regions that facilitate purification may be facilitated by altering the net charge or other function.
  • conservative substitutions are in basic amino acids (arginine, lysine, and histidine), Acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic Substitutions within the family of amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycolic acid, alanine, serine, threonine and methionine). Aromatic acid substitutions which generally do not alter the specific activity are known in the art and are described, for example, by H. Neurath and R. Hill, 1979, in Protein, Academic Press, New York. The most common alternatives are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
  • the present invention also encompasses fusion polypeptides or cleavable fusion polypeptides in which other peptides/polypeptides are fused at the N-terminus and/or C-terminus of the ⁇ -conotoxin peptide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art and include ligating a coding sequence encoding a peptide of the invention with a coding sequence encoding the other peptide/polypeptide such that they are in the same reading frame and the expression of the fusion polypeptide is controlled by the same Promoter and terminator.
  • the polypeptide according to any of the invention, wherein the C-terminus of the polypeptide is amidated can be achieved by artificial chemical synthesis, or it can be achieved by amidation enzyme either intracellularly or extracellularly.
  • the polypeptide according to any one of the invention wherein the first cysteine at the N-terminus of the polypeptide forms a disulfide bond with the third cysteine, and the second cysteine and the second The four cysteines form a disulfide bond; or the first cysteine at the N-terminus of the polypeptide forms a disulfide bond with the fourth cysteine, and the second cysteine and the third a cysteine forms a disulfide bond; the first cysteine at the N-terminus of the polypeptide forms a disulfide bond with the second cysteine, and the third cysteine and the fourth half Cystine forms a disulfide bond.
  • the above polypeptide of the present invention is a conotoxin peptide; specifically, it is an ⁇ -conotoxin peptide.
  • the above conotoxin peptide can be obtained from Conus lividus produced in Hainan, China. Or extract from brocade snail (C l"S ⁇ / ). It is also possible to chemically synthesize an amino acid sequence (for example, the method in Reference Examples 2-(1) to 2-(3)); or by means of genetic recombination Expression of nucleotides (preparation of nucleotide sequences with reference to Examples 1-(1) to 1-(3) or direct synthesis of polypeptides according to the methods of Examples 2-(1) to 2-(3) ), get the peptide. You can also refer to the following method:
  • Another aspect of the invention relates to a method of producing a polypeptide according to any of the inventions, comprising the steps of:
  • a linear polypeptide is synthesized on an ABI Prism 433a peptide synthesizer or by a manual method.
  • the side chain protecting group of Fmoc® acid is: Pmc(Arg), Trt or Acm(Cys), But (Thr, Ser, Tyr), OBut ( Asp) and Boc (Lys);
  • step 2) cutting the linear polypeptide synthesized in step 1) from the resin;
  • a further aspect of the invention relates to a polynucleotide encoding a nucleic acid sequence of a polypeptide of any of the invention.
  • polynucleotide according to any one of the present invention which is or comprises a nucleotide sequence according to any one of (1) to (3) below:
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16-21, SEQ ID NO: 22-25, SEQ ID NO: 29 or a nucleotide sequence shown by any one of SEQ ID NO: 31;
  • a further aspect of the invention relates to a nucleic acid construct comprising the polynucleotide of any of the invention.
  • a further aspect of the invention relates to an expression vector comprising the nucleic acid construct of any of the invention.
  • a further aspect of the invention relates to a transformed cell comprising the expression vector of any of the invention.
  • a further aspect of the invention relates to a fusion protein comprising the polypeptide of any of the invention.
  • a further aspect of the invention relates to a pharmaceutical composition comprising or comprising a fusion protein of the invention; optionally further comprising a pharmaceutically acceptable carrier or adjuvant.
  • a further aspect of the invention relates to a method of blocking an acetylcholine receptor comprising the step of using an effective amount of the polypeptide of any of the invention or the fusion protein of the invention; in particular, the acetylcholine receptor is ⁇ 3 ⁇ 2 Acetylcholine receptor, (X 6/ 0C 3 P 2 P 3 acetylcholine Alkali receptor or (3 ⁇ 43 ⁇ 4 acetylcholine receptor.
  • a further aspect of the invention relates to a method of screening for an acetylcholine receptor inhibitor or determining an acetylcholine receptor subtype, the method comprising: acetylcholine receptor by any of the present invention in the presence or absence of a candidate compound
  • polypeptide or fusion protein eg alpha-conotoxin LvIA/LvD21
  • ⁇ -conotoxin TxIB/Txd4 to specifically block (X 6/ (X 3 P 2 P 3 acetylcholine receptor or (For example, ⁇ -conotoxin TxIC/Txdl) can specifically block ⁇ 3 ⁇ 4 acetylcholine receptor
  • the acetylcholine receptor is ⁇ 3 ⁇ 2 subtype, ( ⁇ 6 ⁇ 2* subtype ((X6/0C3P2P3 acetylcholine receptor) or ⁇ 3 ⁇ 4 sub A acetylcholine receptor of the type.
  • a further aspect of the invention relates to the polypeptide of any of the invention or the fusion protein of the invention for blocking acetylation Use of a choline receptor; specifically, the acetylcholine receptor is an ⁇ 3 ⁇ 2 acetylcholine receptor, (X6/0C3P2P3 acetylcholine receptor or ⁇ 3 ⁇ 4 acetylcholine receptor).
  • a further aspect of the invention relates to the use of a polypeptide according to any of the invention or a fusion protein of the invention for the preparation of a medicament or a reagent for blocking an acetylcholine receptor; in particular, the acetylcholine receptor is an ⁇ 3 ⁇ 2 acetylcholine receptor (X6/0C3P2P3 acetylcholine receptor or ⁇ 3 ⁇ 4 acetylcholine receptor.
  • a further aspect of the invention relates to the polypeptide of any of the invention or the fusion protein of the invention for the preparation of a therapeutic and/or prophylactic and/or adjuvant treatment for a neurological disorder such as addiction, neuralgia, Parkinson's disease, or dementia
  • a neurological disorder such as addiction, neuralgia, Parkinson's disease, or dementia
  • the neuralgia is caused by: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal Neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve spasm, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multi-point motor neuralgia, chronic congenital sensory neuropathy, acute Severe spontaneous neuralgia, crushing neuralgia, vasculitis, blood Tube inflammation, ischemia, uremia, childhood bile liver disease, chronic respiratory disorder, compound neuralgi
  • a further aspect of the invention relates to a therapeutic and/or prophylactic and/or adjunctive treatment of diseases of the nervous system such as pain, alcohol and tobacco addiction, mental retardation, dementia, schizophrenia, central nervous disorders, epilepsy, Parkinson's disease , mental illness, neuromuscular block, myasthenia gravis, depression, high blood pressure, arrhythmia, asthma, muscle relaxation, stroke, breast cancer and lung cancer, etc., or a method of killing pests, analgesics, quitting smoking, or A method of detoxification comprising the step of administering an effective amount of a polypeptide of the present invention (conotoxin peptide or a propeptide thereof) or a pharmaceutical composition of the present invention or a fusion protein of the present invention; specifically, the addiction is caused by nicotine, Morphine, cocaine, alcohol, etc., which can cause addiction; the neuralgia is caused by: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster
  • the conotoxin peptide of the present invention can exert an activity of eliminating addiction and analgesia by binding to ⁇ 3 ⁇ 2 acetylcholine receptor (nAChR), 6/ 3 ⁇ 2 ⁇ 3 acetylcholine receptor or ⁇ 3 ⁇ 4 acetylcholine receptor. It can be applied to research, diagnosis and treatment of addiction, neuralgia, Parkinson's disease, dementia, schizophrenia, depression and other neurological diseases, as well as useful molecular probes for research and other aspects. Different a-CTx have different affinities for vertebral pusher receptors, sometimes differing by several orders of magnitude.
  • nAchR vertebral promoter
  • the polypeptides of the invention are useful in the treatment of various neuralgia.
  • Neuralgia is pain caused by primary or secondary damage or dysfunction or transient disturbance of the surrounding or central nervous system, manifested as spontaneous pain, hyperalgesia, hyperalgesia, and the like.
  • neuralgia including cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve spasm, drug poisoning, industrial pollution Poisoning, lymphatic neuralgia, myeloma, multi-point motor neuralgia, chronic congenital sensory neuropathy, acute strenuous spontaneous neuralgia, crushing neuralgia, vasculitis (vasculitis) / ischemia, uremia, child bile Liver disease, chronic respiratory disorder, compound neuralgia, multiple organ failure, sepsis/sepsis, hepatitis, porphyria, vitamin deficiency, chronic liver disease, primary biliary sclerosis, hyperlipidemia, leprosy, Lyme Arthritis, sensory fasciitis, allergies, etc.
  • Addiction Addiction
  • the polypeptides of the invention are involved in the treatment of various addictions caused by dependent substances.
  • Addiction refers to the periodic or chronic poisoning state of people who repeatedly use psychoactive substances.
  • Psychoactive substances are nicotine, opium, heroin, methamphetamine (ice), morphine, marijuana, cocaine, and other narcotic drugs and psychotropic substances that are regulated by the State and can cause addiction.
  • Addiction is associated with a large number of dopamines produced in the brain. It is manifested by the uncontrollable application of preferred substances and the difficulty of self-made or difficult to correct the use of behavior. For the purpose of obtaining psychoactive substances to achieve good feelings or avoid withdrawal pain, it can be used as a means. Typically, tolerance is increased and withdrawal symptoms often occur after discontinuation of substance use.
  • the invention also relates to a nucleic acid construct comprising a nucleic acid sequence of the invention and one or more regulatory sequences operably linked thereto, which, under compatible conditions, directs the coding sequence in a suitable host cell expression.
  • Expression is understood to include any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • nucleic acid constructs are defined herein as single-stranded or silent-stranded nucleic acid molecules that are isolated from natural genes, or modified to contain nucleic acid fragments that are combined and juxtaposed in an unnatural manner.
  • nucleic acid construct comprises all of the regulatory sequences necessary for expression of a coding sequence of the invention, the term nucleic acid construct is synonymous with an expression cassette.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the acid sequence of its protein product. The boundary of the coding sequence is usually the ribosome binding site immediately upstream of the mRNA 5, open reading frame (for Prokaryotic cells) and transcription termination sequences immediately downstream of the open reading frame of mRNA 3.
  • the coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • the isolated nucleic acid sequence encoding the peptide of the present invention can be manipulated in a variety of ways to express the peptide. It may be desirable or necessary to process the nucleic acid sequence prior to insertion into the vector, depending on the expression vector. Techniques for modifying nucleic acid sequences using recombinant DNA methods are well known in the art.
  • regulatory sequence is defined to include all components necessary or advantageous for expression of a peptide of the invention. Each regulatory sequence may be naturally or foreign to the nucleic acid sequence encoding the polypeptide. These regulatory sequences include, but not Limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, regulatory sequences include promoters as well as transcription and translation termination signals. Sites for ligating a regulatory sequence to the coding region of a nucleic acid sequence encoding a polypeptide can provide a regulatory sequence with a linker.
  • operably linked is defined herein as a conformation wherein the regulatory sequences are located at appropriate positions relative to the coding sequence of the DNA sequence such that the regulatory sequences direct expression of the polypeptide.
  • the control sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence.
  • the promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polypeptide.
  • the promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and heterozygous promoters, which may be derived from extracellular or intracellular encoding homologous or heterologous to the host cell.
  • the gene of the polypeptide may be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence.
  • the promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polypeptide.
  • the promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and heterozygous promoters, which may be
  • the control sequence may also be a suitable transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription.
  • the termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that can function in the host cell of choice can be used in the present invention.
  • the control sequence may also be a suitable leader sequence, i.e., an mRNA untranslated region that is important for translation of the host cell.
  • the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that can function in the host cell of choice can be used in the present invention.
  • the control sequence may also be a signal peptide coding region which encodes an amino acid sequence attached to the amino terminus of the polypeptide to direct the encoded polypeptide into the cell's secretory pathway.
  • the 5' end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region in which the translational reading frame is naturally ligated to the coding region fragment of the secreted polypeptide.
  • the 5' end of the coding region may contain a signal peptide coding region that is foreign to the coding sequence.
  • the native signal peptide coding region can be simply replaced with a foreign signal peptide coding region to enhance polypeptide secretion.
  • any can guide the invention.
  • the control sequence may also be a propeptide coding region that encodes an amino acid sequence at the end of the polypeptide.
  • the resulting polypeptide is referred to as a zymogen or propolypeptide.
  • the propolypeptide is usually inactive,
  • the propeptide can be cleaved from the propolypeptide by catalytic or autocatalytic conversion to a mature active polypeptide.
  • the peptide region is immediately adjacent to the end of the polypeptide, and the signal peptide region is adjacent to the end of the peptide region.
  • regulatory sequences that modulate the expression of the polypeptide depending on the growth of the host cell.
  • regulatory systems are those that respond to chemical or physical stimuli, including in the presence of regulatory compounds, to open or shut down gene expression.
  • Other examples of regulatory sequences are those that enable gene amplification.
  • the nucleic acid sequence encoding the polypeptide should be operably linked to a regulatory sequence.
  • the invention also relates to recombinant expression vectors comprising the nucleic acid sequences, promoters and transcriptional and translational termination signals of the invention.
  • the various nucleic acids and regulatory sequences described above can be joined together to prepare a recombinant expression vector which can include one or more convenient restriction sites for insertion or substitution of a nucleic acid sequence encoding the polypeptide at such sites.
  • the nucleic acid sequence of the present invention can be expressed by inserting a nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector.
  • the coding sequence can be placed in a vector for operably linked to appropriate expression control sequences.
  • the recombinant expression vector can be any vector (e.g., a plasmid or virus) that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence.
  • the choice of vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced.
  • the vector can be a linear or closed loop plasmid.
  • the vector may be an autonomously replicating vector (i.e., a complete structure that exists extrachromosomally and may be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any mechanism to ensure self-replication.
  • the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome.
  • a single vector or plasmid may be employed, or two or more vectors or plasmids, or transposons, which will generally introduce all of the DNA introduced into the genome of the host cell.
  • the vector of the present invention contains one or more selection markers for facilitating selection of transformed cells.
  • a selectable marker is a gene whose product confers resistance to a biocide or virus, resistance to heavy metals, or confers auxotrophic prototrophy and the like.
  • Examples of bacterial selection markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or the resistance markers of antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline.
  • the vector of the present invention comprises an element which enables stable integration of the vector into the host cell genome, or which ensures that the vector autonomously replicates in the cell independently of the cell genome.
  • the vector may also contain an origin of replication enabling the vector to replicate autonomously in the host cell of interest.
  • the origin of replication may be provided with a mutation that renders it temperature sensitive in the host cell (see, e.g., fEhrlich, 1978, Proc. Natl. Acad. Sci. 75: 1433).
  • More than one copy of the nucleic acid sequence of the invention can be inserted into the host cell to increase the yield of the gene product.
  • An increase in the copy number of the nucleic acid sequence can be accomplished by inserting at least one additional copy of the sequence into the genome of the host cell, or by inserting an amplifiable selectable marker with the nucleic acid sequence, by culturing the cell in the presence of a suitable selection reagent, Cells containing an amplified copy of the selectable marker gene, thereby containing the additional copy nucleic acid sequence, are selected.
  • the invention also relates to a recombinant host cell comprising a nucleic acid sequence of the invention useful for recombinant production of a polypeptide.
  • a vector comprising the nucleic acid sequence of the present invention can be introduced into a host cell such that the vector is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector.
  • host cell encompasses any progeny that differs from the parental cell due to mutations that occur during replication. The choice of host cell depends to a large extent on the polypeptide encoding gene and its source.
  • the host cell can be a prokaryotic cell or a eukaryotic cell, such as a bacterial or yeast cell.
  • the vector can be introduced into a host cell by techniques well known to those skilled in the art. Preparation
  • the invention further relates to a method of recombinantly preparing a peptide of the invention, the method comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid encoding the peptide under conditions suitable for the production of the peptide Sequence; and (b) recovering the peptide.
  • cells are cultured in a nutrient medium produced by a suitable polypeptide by methods known in the art. For example, small-scale or large-scale fermentation (including continuous, batch, batchwise or in a shake flask culture, laboratory or industrial fermentor) in a suitable medium, under conditions that permit expression and/or isolation of the polypeptide. Solid state fermentation) to culture cells.
  • the cultivation is carried out in a suitable medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art.
  • Suitable media may be supplied by the supplier or may be prepared with reference to the disclosed compositions (e.g., as described in the catalogue of the American Type Culture Collection). If the polypeptide is secreted into the culture medium, the polypeptide can be recovered directly from the culture medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the polypeptide produced can be recovered by methods known in the art.
  • the polypeptide can be recovered from the culture medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.
  • polypeptides of the invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and Size exclusion chromatography), HPLC, electrophoresis (eg, preparative isoelectric focusing), differential solubility (eg ammonium sulfate precipitation), SDS-PAGE or extraction (see, eg, protein purification, edited by JC Janson and Lars Ryden) , VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and Size exclusion chromatography
  • HPLC e.g., electrophoresis (eg, preparative isoelectric focusing), differential solubility (eg ammonium sulfate precipitation), SDS-PAGE or extraction (see, eg, protein
  • the present invention also relates to an animal or plant cell transformed with the nucleic acid sequence of the present invention, preferably a plant cell such as wheat or corn, which confers a new trait (e.g., insect resistance) to the transformed host.
  • a plant cell such as wheat or corn
  • This can be used to transform animals with the constructs disclosed herein by techniques well known to those skilled in the art. Or realized by plant cells. Method and preparation for controlling pests
  • Control of pests can be achieved by a variety of methods known to those skilled in the art using the conotoxin peptides or polynucleotides of the invention. These methods include, for example, applying a recombinant microorganism to a pest (or their locus), and transforming the plant with a gene encoding the conotoxin peptide of the present invention. Transformation can be carried out by those skilled in the art using conventional techniques. Essential materials for these transformations are disclosed herein, or can be readily obtained by the skilled artisan by other methods.
  • the formulated preparation containing the conotoxin peptide, or a recombinant microorganism comprising the polynucleotide of the present invention can be applied to the soil.
  • the formulated product can also be applied as a seed coating or root treatment or as a complete planting treatment in the late stages of the crop growth cycle.
  • Formulations may include diffusion-thickening adjuvants, stabilizers, other insecticidal additives, or surfactants.
  • the liquid preparation may be water-based or non-aqueous and used in the form of a foam, a gel, a suspension, an emulsifiable concentrate or the like.
  • the ingredients may include a rheological agent, a surfactant, an emulsifier, a dispersant, or a polymer.
  • the concentration of the pesticide will vary widely depending on the nature of the particular formulation, particularly as a concentrate or as a direct use.
  • the pesticide will be present in an amount of at least 1% by weight, and may be 100% by weight.
  • Dry formulations typically have from about 1% to about 95% by weight of the pesticide, while liquid formulations will typically be from about 1% to about 60% by weight of the solids in the liquid phase.
  • the cell-containing preparation will typically contain from about 10 2 to about 10 4 cells/mgo. These formulations will be used in amounts of about 50 mg (liquid or dry) - 1 kg or more per hectare.
  • the formulation can be applied to pest environments such as soil and plants by spraying, spreading, sprinkling, and the like.
  • the invention further relates to a pharmaceutical composition comprising a peptide of the invention and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition can be used for research, diagnosis, alleviation or treatment and addiction, Neuropathic pain, mental retardation, pain, Parkinson's disease, psychosis, depression, myasthenia gravis, cancer, etc.
  • a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the invention is formulated and administered in a pharmaceutically acceptable manner, taking into account the clinical condition of the individual patient, the site of delivery, the method of administration, and the schedule of administration. Arrange and other factors known to the doctor. Therefore, the "effective amount" for the purposes of this document is determined by these considerations.
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the present invention is administered parenterally, orally, intracerebrally, intrathecally, and the like.
  • “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, capsule material or any type of formulation adjunct.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intrathecal and intraarticular injections and infusions.
  • the polypeptide of the present invention can also be administered appropriately by a slow-drying system.
  • the invention also relates to pharmaceutical compositions that specifically block nAChRs.
  • the conotoxin peptide of the present invention can be used as a useful probe for studying the phylogeny of animal nAChRs; as a molecular probe to determine different subtypes of nAChRs; as a molecular model, designing a new drug; as a research, diagnostic neurological Tools and treatments for diseases such as addiction, Parkinson's disease, mobility disorder, schizophrenia, etc.; candidates for the treatment of breast cancer, lung cancer, small cell lung cancer, etc.
  • a peptide insecticide it has been developed as a new type of biopharmaceutical.
  • the ⁇ -conotoxin peptide of the present invention is capable of specifically blocking acetylcholine receptors (nAChRs), and has potent analgesic and withdrawal addictive activity, and an effect of treating diseases such as Parkinson's disease, dementia, schizophrenia, and depression.
  • nAChRs acetylcholine receptors
  • FIG. 1 Alpha-conotoxin LvIA/LvD21 (LvIA) propeptide sequence and its encoded propeptide and mature peptide produced by post-translational modification.
  • the arrow refers to the processing site of the post-translational modification.
  • Inferred Protease Hydrolysis Processing Site 1 (processing 1 ) in Basic Amino Acids After the arginine (R); the C-terminal amidation processing site is indicated by the character shading at the position of the glycine indicated by the arrow, ie, processing 2.
  • the mature peptide C-terminal is next to cysteine (Cys)
  • the first glycine residue is often the processing site for amidation post-translational modification.
  • LvIA/LvD21 The mature peptide produced by amidation from processing site 2 is named LvIA/LvD21 (or LvIA), and the sequence is: GCCSHPACNVDHPEIC# (# indicates C-terminal Amidation).
  • the propeptide region is indicated by italics, the mature peptide is underlined, the cysteine (C) is shown in bold type, and the stop codon is indicated by *.
  • Figure 2 Synthetic linear peptide and mature peptide a-LvIA/LvD21 (SEQ ID NO: 4) sequence and its disulfide linkages ⁇ - ⁇ , II-IV, and their corresponding HPLC chromatograms.
  • Figure 2A shows the synthetic linear peptide sequence, and its free-SH of Cysl and Cys3 and the protecting group S-Availacle (S-acetamidomethyl) on Cys2 and Cys4;
  • Figure 2B shows the mature peptide a- after oxidative folding LvIA/LvD21 sequence, and its I-III, II-IV disulfide linkage;
  • Figure 2C shows the HPLC chromatogram of the linear peptide synthesized in Figure 2A with a retention time of 27.713 min;
  • Figure 2D shows It is a HPLC chromatogram of the oxidized peptide of Figure 2B with a retention time of 27.947 min.
  • Figure 3A shows the effect of 100 nM a-LvIA/LvD21 on the current of ⁇ 3 ⁇ 2 nAChR with a clamping voltage of 70 mV.
  • the "C” in Figure 3A refers to the control current, and the arrow indicates 100 nM a -LvIA/LvD21 After 5 minutes of incubation, the current path of the first Ach pulse ( ⁇ 0 nA ), the time interval between each two current traces is 1 min.
  • Figures 3B, 3C, and 3D are the concentration-dose response curves of a-LvIA/LvD21 to various nAChRs subtypes (11 rats, 2 humans), respectively, and the abscissa is the molar concentration of a-LvIA/LvD21 used ( The logarithm of M) (Log[LvIA/LvD21]M); the ordinate is the percentage of dose response (% Response), which is the ratio of the ratio of acetylcholine receptor current to control current for the corresponding concentration of toxin.
  • Figure 3B shows that a-LvIA/LvD21 highly selective blockade of rat ⁇ 3 ⁇ 2 vs.
  • FIG. 3C shows that a-LvIA/LvD21 pairs other rat neuronal subtypes and mice Concentration dose response curves for muscle-type nAChRs;
  • Figure 3D shows that a-LvIA/LvD21 highly selective blockade of human ⁇ 3 ⁇ 2 vs. «6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs concentration dose response curve.
  • the values in Figure 3 are taken from 3 - Average current of 9 Xenopus oocytes.
  • Figure 4 Current effects of various doses of a-LvIA/LvD21 on various nAChRs.
  • Figure 4A shows the effect of 100 nM a-LvIA/LvD21 on the current of ⁇ 3 ⁇ 2 nAChRs in rats;
  • Figure 4B shows the effect of 10 ⁇ a-LvIA/LvD21 on the current of ⁇ 2 ⁇ 2 nAChRs;
  • Figure 4C 10 ⁇ a-LvIA/LvD21 Current effects of murine muscle type ( ⁇ ) nAChRs.
  • “C” refers to the control current, immediately following "C, followed by the toxin concentration of a-LvIA/LvD21.
  • FIG. 4 shows that 100 nM a-LvIA/LvD21 specifically blocks ⁇ 3 ⁇ 2 nAChR, while 10 ⁇ does not block the ⁇ 2 ⁇ 2 ( ⁇ ) and ⁇ 1 ⁇ ( ⁇ ) nAChRs subtypes at all.
  • Figure 5 Concentration dose response curves of a-LvIA/LvD21 versus ⁇ 3 ⁇ 2 nAChRs and its seven ⁇ 2 mutants.
  • the mutants in Figure 5 ⁇ are (3 ⁇ 43P2[F119Q], ⁇ 3 ⁇ 2[ ⁇ 11 ⁇ ]; the mutants in Figure 5 ⁇ are (3 ⁇ 43P2[F119Q], ⁇ 3 ⁇ 2[ ⁇ 59 ⁇ ], «3p2[T59L]; the mutant in Figure 5C is ⁇ 3 ⁇ 2 [ ⁇ 59 ⁇ ], ⁇ 3 ⁇ 2 [ ⁇ 79 ⁇ ], a3p2[Q34A].
  • Figure 6 shows 10 nM a-LvIA/LvD21 versus rat ⁇ 3 ⁇ 2 nAChRs wild type (A), and mutant «3P2[F119Q] ( ⁇ ), ⁇ 3 ⁇ 2[ ⁇ 59 ⁇ ] (C) and ⁇ 3 ⁇ 2[ ⁇ 11 ⁇ ] (D
  • the current affects the situation and the different elution rates after blocking.
  • Figure 6A 10 nM a-LvIA/LvD21 blocks approximately 50% of the wild type of ⁇ 3 ⁇ 2 nAChRs, eluting at a fast rate and fully recovering within 2 min;
  • Figure 6B 10 nM a-LvIA/LvD21 blocking mutant (3 ⁇ 43P2) [F119Q] All currents, the elution rate is slow, and the current recovers after 12 minutes of elution;
  • Figure 6C 10 nM a-LvIA/LvD21 blocks all the currents of the mutant ⁇ 3 ⁇ 2 [ ⁇ 59 ⁇ ], the elution rate is very slow, wash The current after 20 min was returned to 27% of the control current.
  • Figure 7 Analgesic effect on the CCI model 1 - 24 h after a-LvIA/LvD21 intraperitoneal administration (IP).
  • the negative control Saline is saline (Saline) and the positive control is morphine.
  • Mephine which was administered at a dose of 1 mg/kg rat body weight
  • a-LvIA/LvD21 was administered at a dose of 1 nmol/kg rat body weight.
  • the abscissa Time(hours) is the number of hours after administration; the ordinate Mechanical Threshold is the observed pain threshold and the basic pain threshold.
  • the percentage ratio of (100) (% of basal), the ordinate of each point in the graph is the mean and standard error (Mean ⁇ SD).
  • Figure 8 Analgesic effect on the CCI model 7-14 days after a-LvIA/LvD21 intraperitoneal administration (IP).
  • the negative control Saline is saline (Saline)
  • the positive control is morphine.
  • a-LvIA/LvD21 was administered at a dose of 1 nmol/kg rat body weight.
  • Figure 9 Analgesic effect on a mouse hot plate test model within 120 min after a-LvIA/LvD21 intraventricular administration (ICV).
  • the negative control Saline is saline (Saline)
  • the positive control is morphine (Morphine)
  • the dose is 100 g/kg mouse body weight
  • the a-LvIA/LvD21 dose is 0.1 nmol/kg mouse body weight.
  • the abscissa Time(min) is the number of minutes after administration
  • the ordinate Threshold (sec) is the observed pain threshold in seconds.
  • FIG. 10 (X-conotoxin TxIB/Txd4 (TxIB) propeptide sequence and its encoded propeptide and mature peptide produced by post-translational modification. Arrows refer to the processing site for post-translational modification. Hydrolysis processing site 1 (processing site 1 ) behind the basic amino acid (R ); C-terminal amidation processing sites may be at the position of the two sucrose acids indicated by the arrow, with the bottom of the character The first or second glycine residue at the C-terminus of the mature peptide at the C-terminus of the cysteine (Cys) is often the processing site for the amidated post-translational modification, from the processing site 2 or the processing site.
  • Hydrolysis processing site 1 behind the basic amino acid (R ); C-terminal amidation processing sites may be at the position of the two sucrose acids indicated by the arrow, with the bottom of the character
  • TxIB/Txd4 The mature peptide produced by amidation was named TxIB/Txd4 (or TxIB), and the sequence was: GCCSDPPCRNKHPDLC# (# indicates C-terminal amidation); the mature peptide produced by amidation from processing site 3 was named TxIB/Txd4 ( G) (or TxIB(G)), the sequence is: GCCSDPPCRNKHPDLCG# (# indicates C-terminal amidation).
  • the C-terminus of TxIB(G) is only one more glycine (G) than TxIB and is an analog of TxIB.
  • the propeptide region is indicated by italics, and the mature peptide is underlined, wherein cysteine (C) is shown in bold type and the stop codon is indicated by *.
  • Figure 11 shows the mature peptide sequences of (X-TxIB/Txd4 (Figure 11A) and TxIB (G) ( Figure 11B) and their disulfide linkages I-III, II-IVo
  • Figure 12 (X-TxIB and TxIB(G) are (3 ⁇ 46/a3p2p3iiAChR highly selective specific blockers.
  • Figure 12A shows the ⁇ a -TxIB pair (the current effect of 3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 nAChR.
  • Figure 12A) C" refers to the control current, and the arrow indicates the current trace ( ⁇ 0nA) formed by the first Ach pulse after 5 minutes of incubation with ⁇ a -TxIB.
  • Figure 12B shows (X -TxIB and TxIB (G For the concentration-dose response curve of (3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 nAChR, the abscissa is the logarithm of the molar concentration (M) of oc-TxIB and TxIB(G) used (Log [TxIB and TxIB(G)]M); Coordinates are the percentage of dose response (% Response), which is the ratio of the ratio of acetylcholine receptor current to the control current for the corresponding concentration of toxin.
  • Figure 12C shows the concentration-dose response curve of oc-TxIB for various nAChRs subtypes, (X -TxIB specifically blocks ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChR with a half-blocking dose (IC50) of 28 nM. At 10 ⁇ toxin concentration, TxIB has no blocking effect on other subtypes with an IC50 > 10 ⁇ . Average current taken from 3-5 Xenopus oocytes .
  • Figure 13 shows a 1 ⁇ - ⁇ pair (3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 nAChR (Fig. 13A), and 10 ⁇ - ⁇ is very close to it ( ⁇ 3 ⁇ 2 (Fig. 13B), ⁇ 6/ ⁇ 3 ⁇ 4 (Fig. 13C), oc3P4 (Fig. 13D)
  • C refers to the control current, followed by "C” (X-TxIB toxin concentration.
  • the arrow indicates that TxIB is blocked after 5 minutes of incubation.
  • the current path of the first Ach pulse formed by the subtype of the body.
  • 1 ⁇ - ⁇ specific block (3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 nAChR (Fig. 13A), and 10 ⁇ completely Not blocking ( ⁇ 3 ⁇ 2 (Fig. 13B), (3 ⁇ 46/ ⁇ 3 ⁇ 4 (Fig. 13C) and ( ⁇ 3 ⁇ 4 (Fig. 13D) nAChRs subtype).
  • FIG 14 ⁇ -conotoxin TxIC/Txdl (TxIC) propeptide sequence and its encoded propeptide and mature peptide produced by post-translational modification.
  • the arrow refers to the processing site of the post-translational modification.
  • the inferred protease hydrolysis site 1 (processing site 1 ) is behind the basic amino acid arginine (R ); the C terminal amidation processing site is at the position of the succinic acid indicated by the arrow, with character shading Representation, ie, processing site 2.
  • the first glycine residue at the C-terminus of the mature peptide immediately adjacent to cysteine (Cys) is often the processing site for amidation post-translational modification, and the mature peptide produced by amidation from processing site 2 Named TxIC/Txdl (or TxIC), the sequence is: GCCSHPVCSAMSPIC # (# indicates C-terminal amidation).
  • the propeptide region is indicated by italics, and the mature peptide is underlined, wherein the cysteine (C) is shown in bold type and the stop codon is indicated by *.
  • Figure 15 A shows the mature peptide a-TxIC/Txdl (SEQ ID NO: 28) sequence and its two key linkages ⁇ - ⁇ , II-IV.
  • ⁇ shows the HPLC chromatogram of ⁇ -TxIC/Txdl containing ⁇ - ⁇ , II-IV dichotomous linkage.
  • the chromatographic conditions of the toxin peptide are: Reversed analysis column with Vydac C18 HPLC, within 40 minutes Linear gradient elution, B solution from 15% to 50%, solution A from 85% to 50%, solution A is 0.65% trifluoroacetic acid (TFA), B is 0.5% TFA and 90% acetonitrile ( An aqueous solution of acetonitrile ).
  • the UV analysis wavelength was 214 nm, and the peak time of TxIC, that is, the retention time was 23.366 minutes.
  • Figure 16 Selective strong blocker of ⁇ -TxIC «3 ⁇ 4 nAChR.
  • A shows the effect of ⁇ ⁇ -TxIC on the current of ⁇ 3 ⁇ 4 nAChR.
  • C refers to the control current, and the arrow indicates the current trace ( ⁇ 0 nA ) formed by the first Ach pulse after 5 minutes of incubation with ⁇ ⁇ -TxIC.
  • B shows ⁇ -TxIC
  • the abscissa is the logarithm of the molar concentration (M) of the ⁇ -TxIC used (Log[TxIC]M); the ordinate is the dose response percentage (% Response) Is the ratio of the ratio of acetylcholine receptor current to control current at the corresponding concentration of toxin.
  • ⁇ -TxIC specific blockade (3 ⁇ 43p4 iiAChR, half-blocking dose (IC 50 )) It is only 12.5 nM; ⁇ -TxIC also has a certain blocking effect on ⁇ 6/ ⁇ 3 ⁇ 4 nAChR, and its half-blocking dose (IC 5 ) is 94 nM; ⁇ -TxIC has a weak blocking effect on ⁇ 2 ⁇ 4 nAChR, half of it The blocking dose is up to 4550 nM. At 10 ⁇ toxin concentration, TxIC has no blocking effect on other subtypes, its IC 5 . > 10 M. The values in the figure are the average values of currents taken from 3 - 8 Xenopus oocytes.
  • Figure 17 shows the effect of 1 ⁇ ⁇ -TxIC on ⁇ 3 ⁇ 4 nAChR ( A ), and the current effect of 10 ⁇ ⁇ -TxIC on ⁇ 4 ⁇ 4( ⁇ ), a7(C) nAChRs.
  • C refers to the control current
  • C is the toxin concentration of a-TxIC.
  • the arrow indicates that after 5 minutes of incubation, TxIC blocks the current trace formed by the first Ach pulse of the corresponding receptor subtype.
  • 1 ⁇ ⁇ -TxIC specifically blocks ⁇ 3 ⁇ 4 nAChR, while 10 ⁇ does not block ⁇ 4 ⁇ 4( ⁇ ) and (3 ⁇ 47(C) nAChRs subtypes at all.
  • kit manual also refer to the literature, Zheng Xiaodong, Gao Bingyan, Li Baozhu, Peng Chao, Wu Ai Yin, Zhu Xiaopeng, Chen Xin, Chang Sun Dongting, Russell Lan, Primer Screening for Novel ⁇ -Conotoxin Gene Cloning, Biotechnology, 2011, 21 (4): 40-44.
  • the extracted total DNA of the genomic gland genome was dissolved in 100 L TE, and 5 ⁇ was subjected to 1.0% agarose gel electrophoresis, and the integrity and size of the obtained DNA were detected by using -EcoT14 I digest DNA Marker as a standard.
  • the OD 26 of the DNA solution was determined using a nucleic acid protein analyzer. , OD 28 . Value as well as OD 26 . /OD 28 . Ratio, and calculate the DNA concentration (g'ml- 1 ) purity and DNA yield ( ⁇ ⁇ ⁇ ⁇ ).
  • the extracted DNA was used as a template for the conotoxin gene cloning and used for the following PCR amplification.
  • ⁇ -CTx specific primers were designed.
  • Each primer was an 18 oligonucleotide fragment.
  • the extracted genomic DNA stock solution was diluted and used as a template for PCR amplification.
  • the following PCR amplification system and reaction conditions were used:
  • C lOmin recovers PCR-specific amplification products, which are ligated with T-easy vector (Promega) and transformed into E. coli XL1 strain (other commercial competent E. coli can also be used), and recombination is selected by blue-white colony and ampicillin resistance.
  • the purified recombinant plasmid was extracted for sequencing analysis. The sequencing results are obtained as follows:
  • the italic letters are introns, corresponding to the primers.
  • the sequence of the PCR-specific amplification product obtained was analyzed by DNAStar software, and the encoded protein sequence and the 3,-untranslated region (UTR) sequence were obtained.
  • UTR 3,-untranslated region
  • a novel (3 ⁇ 44 ⁇ -CTx LvIA/LvD21 precursor gene of the present invention, the underlined portion of SEQ ID NO: 1 which encodes the LvIA/LvD21 conotoxin propeptide was obtained.
  • the nucleotide sequence is as follows (114aa):
  • LvIA/LvD21 conotoxin propeptide was presumed to have the amino acid sequence (37aa) shown in SEQ ID NO: 2, hereinafter also referred to as ⁇ -conotoxin LvIA/LvD21.
  • Precursor or - LvIA/LvD21 precursor or LvIA/LvD21 precursor or LvIA or LvD21 precursor as follows:
  • the mature peptide LvIA/LvD21 was further deduced based on the propeptide sequence, which has the amino acid sequence shown in SEQ ID NO: 4 (16 aa; hereinafter also referred to as ⁇ -conotoxin LvIA/LvD21 or - LvIA/LvD21 or LvIA/LvD21 or LvIA Or LvD21):
  • GCCSHPACNVDHPEIC# (SEQ ID NO: 4; # indicates C-terminal acylation).
  • LvIA/LvD21 contains the CC-CC cysteine pattern unique to ⁇ -CTx, disulfide linkages I-III, II-IV (Fig. 2A-B), ie in the first and third cysteine Two pairs of disulfide bonds are formed between, and between the second and fourth cysteines, respectively.
  • LvIA/LvD21 is a 4/7 type ⁇ -CTx (Fig. 1 and Fig. 2A-B).
  • the mature peptide LvIA/LvD21 of the invention may also be obtained by in situ or in vitro processing of the propeptide (SEQ ID NO: 3 or 6) in vivo or in vitro (for example as shown in Figure 1), optionally, in vivo or in vitro.
  • the amidase enzyme amidates its C-terminus.
  • nucleotide sequence encoding the mature peptide of LvIA/LvD21 is as follows (48 bp):
  • the invention also relates to a sequence (17aa) of a mature peptide without a second processing site (processing 2):
  • GCCSHPACNVDHPEIC G SEQ ID NO: 6 ;
  • nucleotide sequence is as follows (54bp): GGATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCA GAAATTTGTGGCTGA (SEQ ID NO: 7); or 51bp
  • the living materials of C. textile Linnaeus collected from the coastal areas such as Hainan Island and Xisha Islands are stored at -80 * € for use.
  • the snail venom gland is first dissected and weighed. Then, the marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., China) was used to extract the genomic DNA of the venom gland. For details, see the kit manual.
  • the extracted total DNA of the snail genome was dissolved in ⁇ TE, and 5 ⁇ was subjected to 1.0% agarose gel electrophoresis, and the integrity and size of the obtained DNA were detected using the -EcoT14 I digest DNA Marker as a standard.
  • the OD260, OD280 value and OD260/OD280 ratio of the DNA solution were determined by a nucleic acid protein analyzer, and the DNA concentration (g.mr 1 ) purity and DNA yield (g*g -1 ) were calculated.
  • the extracted intact DNA was used for the next PCR amplification to template the conotoxin gene clone.
  • Example 1-(1) The method, system, conditions, primers used, and the like of the PCR reaction were referred to in Example 1-(1) except that the template was diluted as the genomic DNA stock extracted in Example 1.
  • the PCR-specific amplification product was recovered and ligated with the T-easy vector (Promega) and transformed into E. coli XL1 strain (other commercial competent E. coli cells can also be used), and recombination was selected using blue-white colonies and ampicillin resistance.
  • the purified recombinant plasmid was extracted for sequencing analysis to obtain a sequence of the PCR-specific amplification product.
  • the sequence of the PCR-specific amplification product obtained was analyzed by DNAStar software, and the encoded protein sequence and the 3, -untranslated region (UTR) sequence were obtained.
  • UTR 3, -untranslated region
  • the TxIB/Txd4 conotoxin propeptide was deduced to have a 41 ⁇ acid-containing protein sequence as shown in SEQ ID NO: 15.
  • TxIB/Txd4 or TxIB/Txd4(G) which has the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, is deduced from the propeptide sequence.
  • the derivation method and principle please refer to Luo. S, Zhangsun D, Zhang B, Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006, 12(ll): 693-704, and Online software ProP 1.0 Server.
  • TxIB/Txd4 or TxIB/Txd4(G) contains the CC-CC cysteine pattern unique to a-CTx, and the disulfide linkage may be I-III, II-IV (Fig. 11, A-B), ie Two pairs of disulfide bonds are formed between the first and third cysteic acids, and between the second and fourth cysteines, respectively.
  • TxIB/Txd4 and TxIB/Txd4(G) are type 4/7 oc-CTx ( Figures 10 and 11).
  • Amino acid sequence (mature peptide) represented by SEQ ID NO: 11 (also referred to herein as oc-conotoxin TxIB/Txd4 or a-TxIB/Txd4 or TxIB/Txd4 or TxIB): GCCSDPPCRNKHPDLC (SEQ ID NO: 11) ) ( 16aa ).
  • the C-terminal cysteine (C) is amidated, i.e., expressed as GCCSDPPCRNKHPDLC #, where # represents C-terminal amidation.
  • SEQ ID NO: 12 also referred to herein as -conotoxin TxIB/Txd4 (G) or oc - TxIB/Txd4 (G) or TxIB/Txd4 (G) or TxIB (G) Sequence (mature peptide):
  • GCCSDPPCRNKHPDLC G (SEQ ID NO: 12) (17aa).
  • the C-terminal glycine (G) is amidated, which is expressed as GCCSDPPCRNKHPDLC G #, where # indicates C-terminal amidation.
  • the C-terminal glycan (position 17) of SEQ ID NO: 12 without amidation may be a recognition site for amidase (intracellular or extracellular), resulting in Amidation of the glycine immediately adjacent to the cysteine (C, position 16), in which case amidated SEQ ID NO: 11 (GCCSDPPCRNKHPDLC # ) will be obtained.
  • GCCSDPPCRNKHPDLC GG (SEQ ID NO: 13) (18aa).
  • the glycine at position 18 of SEQ ID NO: 13 may be the recognition site for the amidase (intracellular or extracellular), resulting in a 17-position of the glycolic acid immediately adjacent to the glycine (G Amidation, in this case amidated SEQ ID NO: 12 (GCCSDPPCRNKHPDLC G # ) will be obtained.
  • the glycine at position 17 of SEQ ID NO: 13 may be a recognition site for amidase (either intracellularly or extracellularly), resulting in amidation of the cysteine (C) at position 16 immediately adjacent to the glycine, In this case, amidated SEQ ID NO: 11 (GCCSDPPCRNKHPDLC#) will be obtained.
  • GCCSDPPCRNKHPDLC GGRR (SEQ ID NO: 14) (20aa).
  • the glycine at position 18 of SEQ ID NO: 14 may be the recognition site for the amidase (either intracellularly or extracellularly), resulting in the 17th position of the glycolic acid immediately adjacent to the glycine (G Amidation, in this case amidated SEQ ID NO: 12 (GCCSDPPCRNKHPDLC G # ) will be obtained.
  • the glycine at position 17 of SEQ ID NO: 14 may be a recognition site for amidase (either intracellularly or extracellularly), resulting in amidation of the cysteine (C) at position 16 adjacent to the glycine, In this case, amidated SEQ ID NO: 11 (GCCSDPPCRNKHPDLC#) will be obtained.
  • SEQ ID NO: 15 also referred to herein as oc-conotoxin TxIB/Txd4
  • amino acid sequence shown by the precursor or oc - TxIB/Txd4 precursor or TxIB/Txd4 precursor or TxIB precursor:
  • the C. textile Linnaeus collected from the coastal areas such as Hainan Island and Xisha Islands were stored at -80. C spare.
  • the snail venom gland is first dissected and weighed. Then, the marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., China) was used to extract the genomic DNA of the venom gland, and the specific operation was carried out according to the kit instructions. The genomic DNA of the venom gland is obtained.
  • the extracted genomic DNA of the venom gland was dissolved in 100 L TE, and 5 ⁇ was subjected to 1.0% agarose gel electrophoresis, and the integrity and size of the obtained DNA were detected by using -EcoT14 I digest DNA Marker as a standard.
  • the OD 26 of the DNA solution was determined using a nucleic acid protein analyzer. , OD 28 . Value as well as OD 26 . /OD 28 . Ratio, and calculate the DNA concentration (g'ml- 1 ) purity and DNA yield ( ⁇ ⁇ ⁇ ⁇ ).
  • the extracted DNA was used as a template for the conotoxin gene cloning and used for the following PCR amplification.
  • the method, system, conditions, primers used, and the like of the PCR reaction are described in Reference Example 1-(1), except that the template is extracted from the genomic DNA stock solution extracted in Example 1.
  • the final concentration is 3, 8 ⁇ l of the amplified product is 1.5%.
  • the size of the amplified product was determined by agarose gel electrophoresis, voltage 90V, electrophoresis for 20 min, and DL2000 DNA Marker as standard.
  • the PCR amplification product is recovered and linked to the T-easy vector (Promega) and transformed into E. coli XL1 strain (other commercial competent E. coli can also be used), using blue and white Colonies and ampicillin resistance were selected for recombinant, and the recombinant plasmid was extracted for sequencing analysis.
  • Two sequencing results were obtained, namely SEQ ID NO: 22 and SEQ ID NO: 23 (Fig. 14, 168 bp), as follows:
  • the TxIC/Txdl conotoxin propeptide has the amino acid sequence (37 aa ) shown in SEQ ID NO: 26 or SEQ ID NO: 27, hereinafter also referred to as ⁇ -conotoxin TxIC/Txdl precursor or - TxIC/Txdl precursor or TxIC/Txdl precursor or TxIC Precursor ) :
  • TxIC/Txdl The mature peptide TxIC/Txdl is deduced from the propeptide sequence, which has the amino acid sequence shown in SEQ ID NO: 28 (hereinafter also referred to as ⁇ -conotoxin TxIC/Txdl or ⁇ -TxIC/Txdl or TxIC/Txdl or TxIC):
  • TxIC/Txdl contains the CC-CC cysteine pattern unique to ⁇ -CTx, disulfide linkages I-III, II-IV (Fig. 15A), ie between the first and third cysteine And two pairs of disulfide bonds are formed between the second and fourth cysteines, respectively.
  • the TxIC/Txdl is a 4/6 type ⁇ -CTx (Fig. 14 and Fig. 15A).
  • TxIC/Txdl is a new ⁇ -conotoxin, and its sequence and activity compared with other ⁇ -CTx are shown in Table 6.
  • the mature peptide TxIC/Txdl of the invention may also be obtained by in vivo or in vitro processing of the propeptide (SEQ ID NO: 26 or 27 or 30) in vivo or in vitro (for example as shown in Figure 14), optionally in vivo or The C-terminus is amidated by an amidase in vitro.
  • the nucleotide sequence encoding the TxIC/Txdl mature peptide is as follows (45 bp):
  • ATCTGT SEQ ID NO: 29.
  • the invention also relates to a sequence (16aa) of a mature peptide without a second processing site (processing 2):
  • the LvIA/LvD21 linear peptide was synthesized by the Fmoc method (Fig. 2B). The specific method is as follows:
  • the resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or a manual synthesis method.
  • cysteine the remaining amino acids are protected with standard side chain protecting groups.
  • the first and third cysteine (Cys) of LvIA/LvD21 are protected by Trt (S-trityl), and the second and fourth cysteine-SH are made of Acm (S-acetamidomethyl).
  • the synthesis steps were as follows: Three isomer linear peptides were synthesized on the ABI Prism 433a peptide synthesizer by Fmoc and FastMoc methods in solid phase synthesis.
  • the side chain protecting groups of Fmoc amino acid are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys).
  • the Fmoc HOBT DCC method, Rink amidated resin and Fmoc amino acid were used, and the synthesis procedure was carried out by referring to the instrument synthesis manual. In order to complete the reaction, the piperidine deprotection and coupling time were appropriately extended, and the refractory amino acid was double-coupled to obtain a resin peptide.
  • reagent K trifluoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5: 7.5 : 5 , W W
  • the purified linear peptide was subjected to purity detection using an analytical HPLC C18 column (Vydac) (Fig. 2C), and HPLC chromatographic analysis was carried out under the same conditions as in the preparation and purification.
  • the flow rate was 0.75 ml/min, and the ⁇ -conotoxin LvIA/LvD21 was used.
  • the peak time of the linear peptide was 27.713 min.
  • the first pair of disulfide bonds were first formed between the two cysteine acids of the Trt protecting group by iron oxidizing potassium oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min). After purification of the monocyclic peptide by reversed phase HPLC C18 column (Vydac), iodine oxidation (10 mM iodine in H 2 0: trifluoroacetic acid: acetonitrile (78:2:20 by volume, 10 min), remove the other two and a half Acm on cystine, and a second pair of disulfide bonds between the two cysteines (Fig. 2B).
  • the bicyclic peptide is purified by reverse phase HPLC C18 column (Vydac), which is obtained according to N
  • the order from end to C was oriented between the corresponding cysteines to form a disulfide-bonded ⁇ -conotoxin.
  • the peak time of LvIA/LvD21 was 27.947 min (Fig. 2D) and was identified as correct by mass spectrometry (MS).
  • HPLC chromatographic conditions were as follows: Reversed analysis column with Vydac C 18 HPLC, linear gradient elution in 40 minutes, B solution from 0-40%, solution A from 100% to 60%, solution A was 0.075% A solution of trifluoroacetic acid (TFA), B 0.05% TFA and 90% acetonitrile in water at a flow rate of 0.75 ml/min.
  • TFA trifluoroacetic acid
  • B 0.05% TFA 90% acetonitrile in water at a flow rate of 0.75 ml/min.
  • the UV analysis wavelength is 214.
  • TxIB and TxIB (G) linear peptides were synthesized by Fmoc method (Fig. 11). The specific method is as follows:
  • the resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or a manual synthesis method.
  • cysteine the remaining amino acids are protected with standard side chain protecting groups.
  • TxIB and TxIB G
  • the first and third cysteine (Cy)-SH are protected with Trt (S-trityl), and the second and fourth cysteic acid-SH are Acm (S- Acetamidomethyl) is protected in pairs.
  • the synthesis steps were as follows: Three isomer linear peptides were synthesized on the ABI Prism 433a peptide synthesizer by Fmoc and FastMoc methods in solid phase synthesis.
  • the side chain protecting groups of Fmoc amino acid are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys).
  • Fmoc HOBT DCC method Rink amidated resin And Fmoc amino acid, the synthesis step is carried out according to the instrument synthesis manual. In order to complete the reaction, the piperidine deprotection and coupling time were appropriately extended, and the refractory amino acid was arbitrarily coupled to obtain a resin peptide.
  • the linear peptide was cleaved from the resin with reagent K (emergenceuoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5 : 7.5: 5,v / v / v / v / v / v) and precipitated and washed with ice diethyl ether
  • the crude linear peptide was recovered and purified by preparative reverse HPLC C18 column (Vydac) eluting with a linear gradient of 2-42% B60, 42-47 min 42-100% B60 in 0-40 min.
  • Solvent B60 is an aqueous solution of 60% ACN (acetonitrile), 40% H20, 0.05% TFA (triHuoroacetic acid); solvent A 0.05% TFA.
  • Purified linear peptide was purified by analytical HPLC C18 column (Vydac) The elution gradient was 0 - 40 min 2 - 42% B60, 42-47 min 42 - 100% B60, and the flow rate was 1 mL/min. It has a purity of over 95% and is used for oxidative folding.
  • the first pair of disulfide bonds were first formed between the two cysteine acids of the Trt protecting group by iron oxidizing potassium oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min).
  • the monocyclic peptide was purified by reversed phase HPLC C18 column (Vydac) and iodine was oxidized (10 mM iodine in H 2 0:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min), and the other two and a half were removed.
  • Acm on the cysteic acid, and a second pair of disulfide bonds between the two cysteines were first formed between the two cysteine acids of the Trt protecting group by iron oxidizing potassium oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min).
  • the monocyclic peptide was purified by reversed phase HPLC C18 column (Vydac) and io
  • the bicyclic peptide is purified by reverse phase HPLC C18 column (Vydac), which is obtained from the N-terminus to the C-terminus.
  • the sequence was oriented between the corresponding cysteine to form a disulfide bond (X-conotoxin) and was identified as correct by mass spectrometry (MS).
  • TxIB and TxIB(G) after oxidation folding are consistent with the measured molecular weight: the theoretical molecular weight of TxIB is 1738.7 Da, the molecular weight of TxIB is 1738.6 Da; the theoretical molecular weight of TxIB (G) is 1795.7 Da; TxIB The molecular weight measured by (G) was 1795.6 Da.
  • the polypeptide concentration was determined by colorimetry at a wavelength of 280 nm, and the polypeptide concentration and mass were calculated according to the Beer-Lambert equation. These quantified folded toxin peptides were used in subsequent activity experiments.
  • Example 2- ⁇ 3) Synthesis of ⁇ -conotoxin TxIC
  • the TxIC linear peptide was synthesized by the Fmoc method (Fig. 15A). The specific method is as follows:
  • Resin peptides are synthesized by Fmoc chemistry, and peptide synthesizers or hands can be used. Synthetic method for synthesizing resin peptides. In addition to cysteine, the remaining amino acids use standard side chain protecting groups. The first and third cysteine (Cys) of TxIC are protected by Trt (S-trityl), and the second and fourth cysteine-SH are protected by Acm (S-acetamidomethyl). . The synthesis procedure was as follows: three isomer linear peptides were synthesized on the ABI Prism 433a polypeptide synthesizer by Fmoc and FastMoc methods in solid phase synthesis.
  • the side chain protecting groups of Fmoc amino acids are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys).
  • Fmoc HOBT DCC method Rink amidated resin and Fmoc
  • the amino acid, synthesis steps are performed in accordance with the instrument synthesis manual. In order to complete the reaction, the piperidine deprotection and coupling time are appropriately extended, and the refractory amino acid is arbitrarily coupled to obtain a resin.
  • the linear peptide was cleaved from the resin with reagent K (trifluoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5 : 7.5: 5 , v / v / v / v / v / v), and precipitated and washed with ice diethyl ether
  • reagent K trifluoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5 : 7.5: 5 , v / v / v / v / v v v v
  • the crude linear peptide was recovered and purified by preparative reverse-HPLC C18 column (Vydac) with a linear gradient of 15 - 50% B90 in 0 - 40 min, 40 - 45 min 50 - 100% B90.
  • Solvent B90 is 90% A
  • UV absorption analysis was performed at 214 nm.
  • the purified linear peptide was subjected to purity analysis using an analytical HPLC C18 column (Vydac) with an elution gradient of 0-40 min 2-42% B60, 42-47 min 42-100% B60, and a flow rate of 1 mL/min. It has a purity of over 95% and is used for oxidative folding.
  • the formation of the first two cysteines of the Trt protecting group was achieved by iron oxidizing potassium oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min). A pair of disulfide bonds.
  • the monocyclic peptide was purified by reversed phase HPLC C18 column (Vydac) and iodine was oxidized (10 mM iodine in H 2 0:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min), and the other two and a half were removed. Acm on the cysteic acid, and a second pair of disulfide bonds between the two cysteines.
  • the bicyclic peptide was purified by reversed phase HPLC C18 column (Vydac), and the linear gradient of elution was still at 0 - 15-50% B90 in 40 min, 40-45 min 50 - 100% B90.
  • Solvent B90 is 90% ACN (acetonitrile), 10% H 2 0, 0.5% TFA (trifluoroacetic acid); Solvent A is 0.65% TFA
  • the aqueous absorption analysis was carried out at a wavelength of 214 nm, that is, ⁇ -conotoxin which was formed to form a disulfide bond between the corresponding cysteines in order from the N-terminus to the C-terminus, and the peak time of TxIC was 23.366.
  • Min Fig. 15B
  • MS mass spectrometry
  • the monoisotopic mass of the oxidized and folded TxIC is consistent with the measured molecular weight: the theoretical molecular weight of TxIC is 1488.81 Da, and the molecular weight of TxIC is 1488.4266 Da, which is 4 Da less than the linear peptide molecular weight of 1492.815 Da.
  • the polypeptide concentration was determined by colorimetry at a wavelength of 280 nm, and the polypeptide concentration and mass were calculated according to the Beer-Lambert equation. These quantified folded toxin peptides were used in the activity experiments in the examples below.
  • nAChRs subtypes ⁇ 3 ⁇ 2, «6/ ⁇ 3 ⁇ 2 ⁇ 3, «6/ ⁇ 3 ⁇ 4, ⁇ 9 ⁇ 10, ⁇ 4 ⁇ 2, ⁇ 4 ⁇ 4, ⁇ 3 ⁇ 4, ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 4, ⁇ 7), human ⁇ 3 ⁇ 2, «6/ ⁇ 3 ⁇ 2 ⁇ 3 , ⁇ 3 ⁇ 4, and cRNA of mouse muscle type nAChRs ( ⁇ ), the concentration of which was measured by OD value at UV 260 nm.
  • frog eggs Solution Xenopus laveis oocytes (frog eggs) were harvested and cRNA was injected into frog eggs with 5 ng cRNA per subunit. Muscle nAChR is injected with 0.5 - 2.5 ng of DNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection and was used for voltage clamp recording of nAChRs within 1-4 days after injection.
  • ND96 perfusate 96.0 mM NaCl, 2.0 mM KCl
  • BSA bovine serum albumin
  • All conotoxin solutions also contained 0.1 mg/ml BSA to reduce non-specific adsorption of toxins, which can be used between perfusion toxins or acetylcholine (ACh) using a switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA). Free switching, and a series of three-way solenoid valves (157 valves, Neptune Research, Northboro, MA) allow free switching between perfusion D96 and ACh.
  • the Ach-gated current is set by the two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) in "slow, clamp, and clamp gain at the maximum (x2000) position.
  • a glass electrode was drilled with a glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) with a diameter of x0.75 mm and filled with 3 M KCl as a voltage and current electrode.
  • the membrane voltage was clamped at -70 mV.
  • the entire system is controlled and recorded by computer.
  • the ACh pulse is the ACh that is automatically perfused with Is every 5 min.
  • the concentration of ACh is 10 ⁇ for the muscle-type nAChRs and the neuronal ⁇ 9 ⁇ 10 nAChRs, and ⁇ 7 for the neuron-type nAChRs. 200 ⁇ , all other subtypes are 100 ⁇ . At least 4 eggs were recorded to express the current response of a subtype to different toxin concentrations, as well as the current trajectory.
  • the measured current data were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • Various parameters related to toxin blocking nAChRs were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • LvIA/LvD21 (prepared in Example 2-(1)) on rat ⁇ 3 ⁇ 2 nAChRs Wait for blocking, and elute faster (Figure 3).
  • LvIA/LvD21 has the strongest blocking activity against ⁇ 3 ⁇ 2 nAChRs, and its half-blocking dose is IC 5 . It is only 8.69 nM with an error range of 6.9 - 11.0 n (Table 1). 100 nM a-LvIA/LvD21 completely blocked the current generated by the opening of Ach-gated rat ⁇ 3 ⁇ 2 nAChRs, which eluted completely within 2 min and its blockade was reversible (Fig. 3A).
  • the blocking activity of LvIA/LvD21 on ⁇ 6/ ⁇ 3 ⁇ 4 nAChRs was second, with a half-blocking dose IC 50 and an error range of 120.9 (86.1-169.8) nM; again ⁇ 3 ⁇ 4 with a half-blocking dose of IC 5 .
  • the error range is 148.4 (103.2-213.2) nM.
  • the blocking activity of LvIA/LvD21 on (3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs is weak, its half-blocking dose IC 5 and error range is 852 (590-1230) nM; it has extremely weak blocking activity on ⁇ 7, ⁇ 2 ⁇ 4,
  • the half-blocking dose IC 5 o and the error range are as high as 3000 (1797-4997) nM and 15520 (11600-20770) nM, respectively.
  • Table 1 The dose response curves of LvIA/LvD21 for various nAChRs subtypes are shown in Figures 3B, 3C, and 3D, respectively.
  • a-LvIA/LvD21 blocked ⁇ 3 ⁇ 2 than blocking (3 ⁇ 46/ ⁇ 3 ⁇ 2 ⁇ 3 activity > 100-fold, up to ⁇ 100-fold in rats, and up to ⁇ 305-fold in humans (Fig. 3 ⁇ , 3D, Table 1)
  • a-LvIA/LvD21 is the first ligand to be found to have the best selectivity and discrimination for ⁇ 3 ⁇ 2 vs. «6/ ⁇ 3 ⁇ 2 ⁇ 3. All of the previously found conotoxins that block ⁇ 3 ⁇ 2 are almost simultaneously Blocking (3 ⁇ 46/a3p2p3 iiAChRs.
  • a-LvIA/LvD21 is a true high-selective new blocker of ⁇ 3 ⁇ 2* vs. ⁇ 6 ⁇ 2* nAChRs that we have discovered for a comprehensive study of the subtype in normal and disease states. The function and significance of the following are of great value.
  • the selective blocking of a-LvIA/LvD21 selectively blocks the ⁇ 3 ⁇ 2 nAChRs from 100 nM a-LvIA/LvD21 to ⁇ 3 ⁇ 2 nAChRs (Fig. 4A), and 10 ⁇ a -LvIA/LvD21 can be seen for the current effects of ⁇ 2 ⁇ 2 (Fig.
  • nAChRs (Fig. 4), 100 nM a-LvIA/LvD21 completely blocks ⁇ 3 ⁇ 2 nAChRs (Fig. 4A), Toxins with a concentration of 100 times higher have no resistance to ⁇ 2 ⁇ 2, and ⁇ nAChRs subtypes. Activity (FIG. 4B-C). Therefore, a-LvIA/LvD21 was discovered by the present inventors, and the novel ⁇ -conotoxin which is highly active against ⁇ 3 ⁇ 2 nAChRs is the first one with the best selectivity and discrimination for ⁇ 3 ⁇ 2 vs. «6/ ⁇ 3 ⁇ 2 ⁇ 3. body. Table 1: a-LvIA / LvD21 of various nAChRs IC 5 subtype. Slope of dose response curve
  • a Numbers in parentheses are 95% confidence intervals; a in the table is a 95% confidence interval.
  • b nAChR subtype IC 5 . / ⁇ 3 ⁇ 2 IC 5 .
  • b is the ratio of other subtypes to the a3 ⁇ 2 nAChRs half-blocking dose (IC50).
  • c nAChR subtype IC50 I Human ⁇ 3 ⁇ 2 IC50; c is the human ⁇ 6/3 ⁇ 2 ⁇ 3 subtype and human ⁇ 3 ⁇ 2 nAChRs half resistance Broken dose The ratio of ( IC50 ).
  • Example 3-(2) (X-conotoxin ⁇ and TxIB (G) specific blockade (X 6/ (X 3 ⁇ 2 ⁇ 3 nAChRs experiment)
  • Xenopus laveis oocytes were dissected and cRNA was injected into frog eggs, and each subunit was injected in 5 ng cRNA. Muscle nAChR is injected with 0.5-2.5 ng of DNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection and was used for voltage clamp recording of nAChRs within 1-4 days after injection.
  • ND96 perfusate (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 5 mM HEPES, pH 7.1-7.5) or ND96 (ND96A) with 1 mM atropine at a flow rate of 1 ml/min .
  • BSA bovine serum Albumin
  • All conotoxin solutions also contained 0.1 mg/ml BSA to reduce non-specific adsorption of toxins, which can be used between perfusion toxins or acetylcholine (ACh) using a switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA). Free switching, and a series of three-way solenoid valves (157 valves, Neptune Research, Northboro, MA) allow free switching between perfusion D96 and ACh.
  • the Ach gated current is set by the two-electrode voltage clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) in the "slow” clamp, and the clamp gain is recorded online at the maximum (x 2000) position.
  • the glass electrode was drawn with a 1 mm outer diameter X 0.75 inner diameter mm glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) and filled with 3 M KCl as the voltage and current electrodes.
  • the membrane voltage was clamped at -70 mV.
  • the entire system was controlled and recorded by the computer.
  • the ACh pulse is an ACh that is automatically perfused with Is every 5 minutes.
  • the concentrations of ACh are muscle-type nAChRs and neurotypes (X 9 (X 10 nAChRs eggs are 10 ⁇ ; expression of neuronal nAChRs (X 7 is 200 ⁇ , and other subtypes are 100 ⁇ ) Record at least 4 eggs expressing the current response of a subtype to different toxin concentrations, as well as the current trajectory.
  • the measured current data were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • Various parameters related to toxin blocking nAChRs were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • Table 2 a -TxIB and TxIB(G) for various nAChRs
  • a is a 95% confidence interval.
  • b is the ratio of TxIB(G) to TxIB half-blocking dose (IC50).
  • c is not blocked at 10 ⁇ .
  • a-TxIB/Txd4 ⁇ 6/3 ⁇ 2 ⁇ 3 nAChR is extremely high. From 1 ⁇ a -TxIB/Txd4 pair ( ⁇ 6/( ⁇ 3 ⁇ 2 ⁇ 3 nAChR , and 10 ⁇ -TxIB/Txd4 is very close to it ( ⁇ 3 ⁇ 2 ( ⁇ ) , «6/a3p4(C), The current effect of 3 ⁇ 4(D) nAChRs can be seen (Fig. 13), 1 ⁇ oc -TxIB/Txd4 special ⁇ - ⁇ .3 ⁇ 4 ⁇ 6/ 3 ⁇ 2 ⁇ 3 nAChR (Fig.
  • nAChRs subtype 3 ⁇ 4 have any blocking activity.
  • Human 6/3 ⁇ 2 ⁇ 3 nAChR, -TxIB and TxIB (G) have similar blocking activities as rat nAChR. Therefore, a-TxIB is currently found, and the ⁇ -conotoxin, which is the most selective for nAChR, is shown in Table 3 below.
  • TxIB/Txd4 and TxIB/Txd4(G) compared to other oc-CTx are shown in Table 3.
  • Example 3- ⁇ 3) ⁇ -conotoxin TxIC specifically blocks ⁇ 3 ⁇ 4 and ⁇ 6/ ⁇ 3 ⁇ 4 nAChRs experiments
  • Xenopus laveis oocytes were dissected and cRNA was injected into frog eggs with 5 ng cRNA per subunit. Muscle nAChR is injected with 0.5 - 2.5 ng of DNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection and was used for voltage clamp recording of nAChRs within 1-4 days after injection.
  • ND96 perfusate (96.0 mM NaCl, 2.0 mM KC1) containing 0.1 mg/ml BSA (bovine serum albumin) was gravity-infused.
  • BSA bovine serum albumin
  • All conotoxin solutions also contain 0.1 mg/ml BSA to reduce non-specific adsorption of toxins, which can be used to infuse toxins or ugly with a switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) Free switching between choline (ACh) and a series of three-way solenoid valves (model valves 161T031, Neptune Research, Northboro, MA) allow for free switching between perfusion D96 and ACh.
  • the Ach-gated current is set by the two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) in "slow, clamp, and clamp gain at the maximum (x2000) position.
  • a glass electrode was drilled with a glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) with a diameter of x0.75 mm and filled with 3 M KCl as a voltage and current electrode.
  • the membrane voltage was clamped at -70 mV.
  • the entire system is controlled and recorded by computer.
  • the ACh pulse is the ACh that is automatically perfused with Is every 5 min.
  • the concentration of ACh is that the muscle expression of nAChRs and the neuronal a9al0 nAChRs eggs are ⁇ ; the expression of the neuronal nAChRs ⁇ 7 is 200 ⁇ , all other subtypes are 100 ⁇ .
  • At least 4 eggs were recorded to express the current response of a subtype to different toxin concentrations, as well as the current trajectory.
  • the measured current data were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • Various parameters related to toxin blocking nAChRs were statistically analyzed using GraphPad Prism software (San Diego, CA), and a dose response curve was plotted to calculate the half-block concentration IC 5 of conotoxin.
  • TxIC prepared in Example 2-(3) specifically blocked rat ⁇ 3 ⁇ 4 nAChR and eluted faster (Fig. 16).
  • TxIC is the most potent blocker of ⁇ 3 ⁇ 4 nAChR found to date, with a half-blocking dose of IC 5 . Only 12.5 nM, compared to the activity of other known conotoxins, see Table 4 below.
  • TxIC has the strongest blocking activity against ⁇ 3 ⁇ 4 nAChR, and its half-blocking dose is IC 5 .
  • the error range is 12.5 nM (9.4 - 16.5 nM); TxIC has the next blocking activity for ⁇ 6/ ⁇ 3 ⁇ 4 nAChR, and its half-blocking dose IC 50 and error range is 94.1 nM (73 - 121 nM); TxIC vs ⁇ 2 ⁇ 4 nAChR f Gen weak blocking activity, half-blocking dose IC 5 () and the error in the range of 4550 nM (3950-5230 nM) slope (Hillslope) e TxIC their dose response curves and the error range are: ⁇ 3 ⁇ 4 nAChR , 0.19 (0.66 - 1.44); «6/ ⁇ 3 ⁇ 4 nAChR, 0.26 (0.73 - 1.87); ⁇ 2 ⁇ 4 nAChR, 0.20 (1.48 - 2.42).
  • ⁇ -TxIC has no blocking activity against other nAChRs subtypes, including ⁇ 4 ⁇ 4, ( ⁇ 4 ⁇ 2, ( ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3, ⁇ 2 ⁇ 2, ( ⁇ 9 ⁇ 10, ⁇ 7, ⁇ , ⁇ 5 ⁇ 10 ⁇ (Fig. 16 ⁇ , Table 5))
  • ⁇ -TxIC blocked ⁇ 3 ⁇ 4 by 7.5 times more potent than 3 ⁇ 46/ ⁇ 3 ⁇ 4 and 524 times more strongly than ⁇ 2 ⁇ 4 (Fig. 16B, Table 5).
  • ⁇ -TxIC/Txdl has a high selectivity for blocking ⁇ 3 ⁇ 4 nAChR. From 1 ⁇ ⁇ -TxIC/Txdl to ⁇ 3 ⁇ 4 nAChR, and 10 ⁇ ⁇ -TxIC/Txdl to its very close ⁇ 4 ⁇ 4 ( ⁇ ), (3 ⁇ 47(C) nAChRs can be seen (Figure 17), 1 ⁇ ⁇ -TxIC/Txdl specifically blocks ⁇ 3 ⁇ 4 nAChR (Fig. 17A), whereas the 10-fold higher concentration of toxin has no blocking activity on ⁇ 4 ⁇ 4 (Fig. 17B) and ⁇ 7 (Fig. 17C) nAChRs subtype. For human ⁇ 3 ⁇ 4 nAChR, ⁇ -TxIC has a blocking activity similar to that of rat ⁇ 3 ⁇ 4 nAChR.
  • ⁇ -TxIC is currently the most active ⁇ -conotoxin active against ⁇ 3 ⁇ 4 nAChR, and also has a strong blocking activity on (3 ⁇ 46/a3p4 iiAChR), and its activity is shown in Table 4 below.
  • - a is a 95% confidence interval; b is the ratio of other subtypes to ⁇ 3 ⁇ 4 nAChR half (ICSQ); c is no blocking activity at 10 ⁇ .
  • ⁇ 3 ⁇ 4, (3 ⁇ 46/a3p4 iiAChRs are treatments for neuropsychiatric disorders such as nicotine, morphine and cocaine addiction, neuralgia, Parkinson's disease, dementia, schizophrenia, depression, fear, etc.
  • the drug action target see related literature in the background art. Therefore, the novel ⁇ -conotoxin TxIC/Txdl of the present invention has extremely high application value in the mechanism research, diagnosis and treatment of the above diseases. : ⁇ -conotoxin LvIA/LvD21 blocks the ⁇ 3 ⁇ 2 nAChRs mutant
  • ⁇ -CTx LvIA/LvD21 7 ⁇ 2 mutants of ⁇ 3 ⁇ 2 nAChR ⁇ 3 ⁇ 2[ ⁇ 59 ⁇ ] , ⁇ 3 ⁇ 2[ ⁇ 59 ⁇ ] , ⁇ 3 ⁇ 2[ ⁇ 59 ⁇ ] , ⁇ 3 ⁇ 2 [Villi] , «3p2[F119Q], ⁇ 3 ⁇ 2 [Q34A] , ⁇ 3 ⁇ 2 [ ⁇ 79 ⁇
  • the blocking effect is quite different (Table 6-7; Figure 5-6).
  • These 7 mutants are the key amino acids that bind the ligand to the ⁇ 2 subunit of nAChR. The residue is mutated to the corresponding acid residue in the ⁇ 4 subunit (including ⁇ -CTx MII).
  • ⁇ -CTx LvIA/LvD21 has the least blocking activity against the mutant ⁇ 3 ⁇ 2 [ ⁇ 11 ⁇ ], and its IC 5 .
  • its activity was 8.7-fold lower than that of wild-type ⁇ 3 ⁇ 2 nAChR (IC 50 of 14.5 nM).
  • the blocking activity against mutant a3p2 [F119Q], ⁇ 3 ⁇ 2 [ ⁇ 59 ⁇ ], ⁇ 3 ⁇ 2 [T59L] is very strong, and its IC 5 .
  • the blocking activity of ⁇ -CTx LvIA/LvD21 against mutant (3 ⁇ 43P2[F119Q] is 217 times that of ⁇ 3 ⁇ 2[ ⁇ 11 ⁇ ].
  • MII, LtIA and other previously discovered ⁇ -CTxs to bind ⁇ 3 ⁇ 2 nAChRs. It is different.
  • a is the 95% confidence interval
  • b is the ratio of ⁇ 3 ⁇ 2 nAChRs mutant to wild-type half-blocking dose (IC 5() );
  • c ⁇ 3 ⁇ 2 nAChRs other mutant, wild type and mutant (3 ⁇ 43P2[F119Q] half The ratio of the blocking dose (IC 5 .).
  • ⁇ -CTx LvIA/LvD21 not only had a significant effect on the blocking activity ( IC 50 ) of some ⁇ 3 ⁇ 2 nAChRs mutants, but also had a significant effect on their elution rate (Fig. 6 and Table 7).
  • the results showed that 10 nM a-LvIA/LvD21 blocked about 50% of the wild type of ⁇ 3 ⁇ 2 nAChRs, and the elution rate was fast, and the current completely recovered within 2 min (Fig. 6A); 10 nM a-LvIA/LvD21 blocked The total current of the mutant a3p2[F119Q] was slow, and the current was recovered after 12 minutes of elution (Fig. 6B).
  • the elution rate is relatively slow, it takes 5-8 minutes to elute, and the current can be restored to the control level.
  • the mutant type (3 ⁇ 43P2[F119Q] the elution rate) It is slower and requires 10 - 12 min of elution, and its current can be restored to the control level.
  • the mutant ⁇ 3 ⁇ 2 [ ⁇ 59 ⁇ ] the elution rate is the slowest, and 10 nM LvIA/LvD21 completely blocks its current. The elution at 20 min can only recover to 28 ⁇ 3.5% of the control current.
  • Example 5 Analgesic activity test of a-LvIA/LvD21
  • the Chronic Constriction Injury model was made in SD (Sprague Dawley) rats, and the conotoxin was measured on a nerve using a pressure pain tester (rat 800G, model IITC 2391). Painful analgesic activity.
  • SD (Sprague Dawley) rats were purchased from the Guangdong Medical Laboratory Animal Center.
  • the production of the CCI model is based on the method of Bennett et al. (Bennett G J, Xie Y K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man [J]. Pain, 1988, 33(1): 87).
  • Rats were placed in cages before tube placement, and they were housed in single cages after tube placement.
  • the primary selected rats were divided into five groups according to the random number table, namely the saline negative control group, the morphine positive control group, and the toxin peptide a-LvIA/LvD21 experimental group, wherein the toxin peptide a-LvIA/LvD21 experimental group was repeated 2 Times (ie, the toxin peptide a-LvIA/LvD21 experimental group was used for 3 times).
  • Mechanical pain stimuli were measured before and after surgery, three days, one week, and two weeks after surgery.
  • the tested sciatic nerve chronic crush injury model (CCI) model was used as a whole animal model to test the efficacy of LvIA/LvD21 on neuralgia.
  • the analgesic effect of LvIA/LvD21 on the CCI model was determined by intraperitoneal administration.
  • Saline was used as a blank control, that is, a negative control; morphine (Morphine) was used as a positive control at a dose of 1 mg/kg of rat body weight.
  • the analgesic activity is expressed as Mechanical Threshold, which is the ratio of the observed pain threshold to the underlying pain threshold (100) (% of basal). The larger the value, the better the analgesic effect.
  • Figure 7 shows the analgesic effect of the a-LvIA/LvD21 on the CCI model 1-24 hours after intraperitoneal administration (IP).
  • LvIA/LvD21 showed a strong analgesic effect on neuralgia 1 hour after administration, while the positive control morphine did not have analgesic activity 1 hour after administration; 3 hours after LvIA/LvD21 administration, the town of neuralgia The highest painful effect was achieved, with an average analgesia of 160%, some as high as 200%, while the positive control analgesia of the positive control morphine at 3 hours after administration was 120%; at 24 hours after the LvIA/LvD21 analgesia The value is still much higher than morphine ( Figure 7).
  • the analgesic value was measured within one week after the withdrawal (days 7 - 14), and the results are shown in Fig. 8.
  • the threshold of mechanical pain stimuli in LvIA/LvD21 was significantly higher than that in the morphine group at 7-14 days, and the analgesic effect was best on the 12th day.
  • the average analgesic value reached 200%.
  • the morphine group and the saline control group There was almost no difference in the analgesic values with significant differences. This indicates that the analgesic effect of morphine after discontinuation of administration disappeared, and the analgesic effect of LvIA/LvD21 after discontinuation of administration was sustained (Fig. 8), indicating that LvIA/LvD21 not only analgesic effects on neuralgia, It also has a healing effect.
  • LvIA/LvD21 has a stronger analgesic effect than morphine.
  • the LvIA/LvD21 analgesic effect is 823 - 1176 times stronger than morphine in the CCI model.
  • the analgesic effect of intraperitoneal injection of LvIA/LvD21 on the rat CCI model is strong and has good persistence. Conotoxin itself does not cause addiction.
  • mice with a response latency of less than 5 s or greater than 30 s were excluded from the experiment, and 50 female Kunming mice weighing 18 ⁇ 2 g were selected. Before administration, the mice were placed on a metal plate of a hot plate pain tester (model: USTC 39) at 55 ⁇ 0.5 °C. The latency should be calculated (S).
  • the negative control saline (Saline), the positive control morphine (Morphine), and the ⁇ -conotoxin LvIA/LvD21 were divided into 3 groups, 10 in each group. Each group was administered in the lateral ventricle, and the injection volume was 10 mice.
  • the positive control morphine was administered at a dose of 100 g/kg of mouse body weight; the a-LvIA/LvD21 dose was 0.1 nmol/kg ( ⁇ 0.17 g/kg) of mouse body weight.
  • the positive control morphine was administered 588 times the LvIA/LvD21 at the same weight dose.
  • the mice were placed at 55 ⁇ 0.5 before administration.
  • C's hot plate pain tester (model USTC 39) metal plate, the latency of the mouse hind foot reaction or jump response as a pain threshold, in seconds (s). Each mouse was measured twice as the basal pain threshold, and the second measurement interval was 5 min. In order to prevent burns on the foot, set 60s as the cut-off time. For those over 60s, the pain threshold is 60s. The values were taken at 15, 30, 45, 60, 90, and 120 min after administration as pain thresholds after administration, and the results were expressed as ⁇ 8.
  • ⁇ -CTx LvIA/LvD21 showed strong analgesic activity on the hot plate test model (Fig. 9).
  • the basal pain thresholds of the three groups before the administration were all around 14 - 17s.
  • the pain threshold of the negative control saline (Saline) remained at around 14 - 17 s at all time points.
  • the pain threshold of LvIA/LvD21 rapidly increased to 30 s, morphine pain.
  • the threshold was also rapidly increased to 32 s (Fig. 9).
  • LvIA/LvD21 showed strong analgesic activity, indicating that the analgesic activity of LvIA/LvD21LvIA was very effective.
  • the pain threshold of LvIA/LvD21 decreased slightly, while the pain threshold of morphine continued to decrease.
  • the pain threshold of LvIA/LvD21 was increased by 1.3 - 1.5 compared with morphine. Times. At 120 min after dosing, the pain threshold of LvIA/LvD21 decreased slightly, but it was still 1.3 times higher than the pain threshold at the time of morphine administration.
  • the analgesic effect of LvIA/LvD21 was 764-882 times stronger than morphine on the hot plate model, calculated by the same weight dose.

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Abstract

提供了一种新的α-芋螺毒素肽、其药物组合物及用途。还提供了所述芋螺毒素肽的前肽、其核酸构建体、其表达载体和转化的细胞及其融合蛋白。公开了一种阻断乙酰胆碱受体的方法，以及所述芋螺毒素肽的制药用途。公开的α-芋螺毒素肽能够特异地阻断乙酰胆碱受体（nAChRs，例如α3β2 nAChRs，α6/α3β2β3 nAChR或α3β4 nAChR或α6/α3β4 nAChR）；具有治疗神经痛、成瘾、帕金森症、痴呆、精神分裂症、癌症等疾病的活性；可用于制备镇痛与戒烟戒毒药物、有关精神疾病与癌症治疗药物，以及神经科学工具药等。

Description

α -芋螺毒素肽、 其药物组合物及用途 技术领域
本发明属于生物化学和分子生物学领域， 涉及一种新的 α-芋螺毒 素肽、 其药物組合物、 其制备方法及用途。 本发明还涉及所述芋螺毒 素肽的前肽、 其核酸构建体、 其表达载体和转化的细胞、 以及其融合 蛋白。 本发明还涉及一种阻断乙酰胆碱受体的方法、 以及所述芋螺毒 素肽的制药用途。 背景技术
生活在热带海洋中的肉食性软体动物芋螺（Comis )分泌的芋螺毒 素或芋螺肽（Conotoxin, Conopeptide, CTx ) ， 具有调节各种离子通 道的特殊功能， 在临床上已显示出了重要价值。 芋螺毒素通常含有 10 - 46个氨基酸， 富含二硫键， 生物活性强， 能特异地作用于动物细胞膜 上的受体和离子通道。 尤其是对电压门控或配体门控离子通道（包括少 数 G-蛋白相关受体等 )具有较高的选择性。芋螺毒素按其前体蛋白的内 质网信号肽序列的相似性， 以及半胱氨酸模式， 分为不同的基因家族， 至今， 所有已知的芋螺毒素可分为 19个超家族， 分别为 A、 B、 C、 D、 S、 M、 II、 12、 13、 J、 L、 01、 02、 03、 P、 T、 V、 Y、 K ( Sulan Luo, Sean Christensen, Dongting Zhangsun, Yong Wu, Yuanyan Hu, Xiaopeng Zhu, Sandeep Chhabra, Raymond S. Norton, and J. Michael Mcintosh. A Novel Inhibitor of α9α10 Nicotinic Acetylcholine Receptors from Conus vexillum Delineates a New Conotoxin Superfamily. PLoS ONE, (2013) 8(1): e54648 (1-10)； 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] ; Ye M, Khoo KK, Xu S, Zhou M, Boonyalai N, Perugini MA, Shao X， Chi C, Galea CA, Wang C & Norton RS. A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily. Journal of Biological Chemistry (2012) 287, 14973-14983 ) 。 芋螺毒素按其受体靶位可分为 ou ω、 μ、 δ等多种药 理学家族。 每个超家族根据受体靶类型， 又可分为 α、 αΑ、 κΑ ( Α- 超家族） ， ω、 δ、 κ：、 μθ ( Ο-超家族） ， μ、 ψ、 KM ( M-超家族）等 家族（亚型） 。 其中， α-芋螺毒素是目前发现的选择性最好的烟碱乙酰胆碱受体 (nAChRs)亚型特异阻断剂。 因此， α-芋螺毒素及其作用靶点 nAChRs 在多种疾病机理的研究， 以及药物研发方面具有极其重要的价值。 α-芋 螺毒素是人们最早发现的一类芋螺毒素，通常分子质量较小，一般由 12
- 19个氨基酸残基組成，富含二硫键。 a-芋螺毒素种类繁多，活性多样， 结构变化复杂。 通过其高度保守的信号肽序列、 药理学活性及半胱氨酸 模式可对 a-芋螺毒素进行分类。 a-芋螺毒素的半胱氨酸模式为 CC-C-C, 其中天然肽的二硫键连接方式为 C1-C3 与 C2-C4, 称为球形异构体
( globular isomer )， 二硫键间形成 2个 loop环。 含有 4个半胱氨酸的 α-芋螺毒素线性肽氧化折叠后往往产生 3 种异构体， 除了 C1-C3 与 C2-C4间的天然肽二硫键连接方式（球形异构体）外， 另外两种异构体 分别是带状异构体（ ribbon isomer )与 珠子状异构体 ( bead isomer )。 带状异构体的二硫键连接方式为 C1-C4与 C2-C3;珠子状异构体的二硫 键连接方式为 C1-C2与 C3-C4。球形异构体具有完全的生物活性， 带状 异构体有时通过不同的作用机制也发挥生物活性，珠子状异构体活性往 往减小。二硫键间形成 2个 loop环，根据二三及三四半胱氨酸间氛基酸 数量不同又可把 α-芋螺毒素分为 α3/5， «4/7, «4/6, α4/4和 α4/3等多种 亚家族，每个 loop的特征和残基組成的不同是毒素作用于不同受体亚型 的^ fili ( Ulens C, Hogg RC, Celie PH, et al. Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP[J】. Proc Natl Acad Sci USA 2006; 103: 3615-20； Mcintosh, J. Μ·; Santos, A. D.; Olivera, B. M.， Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. Annual review of biochemistry 1999,65, 59-88； Terlau, H.; Olivera, B. M.， Conus venoms: a rich source of novel ion channel-targeted peptides. Physiological reviews 2004,^/ (1)， 41-68. Gehrmann J, Alewood PF, Craik DJ. Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability. J Mol Biol. 1998, 278(2):401-15; Grishin AA， Wang CI, Muttenthaler M, Alewood PF, Lewis RJ, Adams DJ. Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of alpha3beta4 nicotinic acetylcholine receptors. J Biol Chem. 2010， 285 (29): 22254-63 ) 。
烟碱乙酰胆碱受体 (nAChRs)是动物界普遍存在的具有重要生理 作用和临床研究意义的细胞膜蛋白， 是人类最早发现的一类受体， 可 分为两类： 肌肉型乙酰胆碱受体和神经型乙酰胆碱受体。 nAChRs是细 胞膜上的变构膜蛋白， 介导众多中枢和外周神经系统的生理功能， 包 括学习、 记忆、 成瘾、 应答、 镇痛和运动控制等。 nAChRs激活多巴 胺、 去甲腎上腺素、 五羟色胺、 γ-氛基丁酸等多种神经递质的幹放。 已证实 nAChRs是筛选诊断和治疗一大类重要疾病药物的关键靶点， 这些疾病包括疼痛、 烟酒和毒品成瘾、 智障、 痴呆、 精神分裂症、 中 枢神经紊乱、癫痫症、 帕金森病、精神病、神经肌肉阻滞、重症肌无力、 抑郁症、 高血压、 心率不齐、 哮喘、 肌肉松弛、 中风、 乳腺癌和肺癌等。 至今对于上述疾病还没有对症治疗的药物。 常用的非选择性的 nAChRs激动剂如烟碱， 虽然可以緩解上述神经疾病的症状， 但它们 对心脏和胃肠道产生强烈的副作用， 且有成瘾性。 因此， 开发针对 nAChRs各种亚型具有高选择性的配体药物是治疗上述疾病的关键所 在 （ Livett BG, 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; Taly A, Corringer PJ， Guedin D, Lestage P, Changeux JP. Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov. 2009, 8(9): 733-50； Lay la A,McIntosh JM. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors [J] . Acta Pharmacol Sin 2009 Jun; 30 (6): 771-783. ) 。
然而，要开发这样的药物的前提是，要获得可以特异结合 nAChRs 各种亚型的选择性化合物， 作为工具药来研究和鉴定各种亚型的精细 組成和生理功能， 或直接作为相关疾病的治疗药物。 另外， 在乳腺癌 与小细胞肺癌中，
Figure imgf000005_0001
的激活促进肿瘤细 胞增殖， 用药物阻断这些受体的激活， 可有效地进行早期诊断， 或治 疗这些灾难性癌症。
nAChRs由不同的 α和 β亚基組装成 f艮多种亚型， 每种亚型都有 截然不同的药理学特征。其中肌肉型乙酰胆碱受体由 5个亚^成，含 2个 αΐ亚基， 1个 β亚基， 1个 δ亚基和 1个 γ或 ε亚基， γ或 ε亚基 取决于其是否为胎儿或成体的乙酰胆碱受体。 哺乳动物神经型 nAChRs 亚型比肌肉型 nAChRs复杂得多， 至少有 8种 α亚基、 3种 β亚基， 分 别为 al-a，、 α9、 αΐθ (在雏鸡中存在 α8 ) 、 以及 β2-β4。 其中 «2, «3 和 α4可以分别同 β2或者 β4结合，形成功能性受体， 比如 α2β2、 α3β2、 α2β4等； α9与 αΐθ结合形成功能性受体 α9α10 nAChRs。 此外， α7和 «9可以形成同源多聚体。 由于缺乏针对各种亚型的高选择性配体化合 物， 要研究和阐明各种各样的 nAChRs亚型的精细结构和功能面临诸 多挑战。
药物成瘾既是医疗难题亦^ 重的社会问题。
Figure imgf000005_0002
碱（尼古丁） 引起的， 其体内受体就是烟碱乙酰胆碱受体 ( nAChRs )
( Azam L, Mcintosh JM. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol Sin. 2009;
30(6): 771-783 ) 。 研究表明， 表 i½多巴胺能 (DA)神经元的 nAChRs 是治疗神经精神疾病， 如烟碱、 吗啡与可卡因等的成瘾、 帕金森病、 痴 呆、 精神分裂症、 抑郁等的药物作用靶点（Larsson, A.; Jerlhag, E.; Svensson, L.; Soderpalm, B.; Engel, J. A., Is an alpha-conotoxin Mil-sensitive mechanism involved in the neurochemical, stimulatory, and rewarding effects of ethanol? Alcohol 2004,34 (2-3), 239-50. Jerlhag, E.; Egecioglu, E.; Dickson, S. L.; Svensson, L.; Engel, J. A., Alpha-conotoxin Mil-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus accumbens. European neuropsychopharmacology, 2008,18 (7)， 508-18)。 阻断 α3β2和 α6β2* nAChRs 的 α-芋螺毒素 Mil可部分和区别性地阻断纹状体中烟碱引起 的多巴胺幹放， 突触前的 nAChRs至少包含 2类亚型， 即 Mil-敏感型 与 Mil-不敏感型，调控多巴胺神经元的 DA幹放（ Kaiser SA, Soliakov L, Harvey SC, Luetje CW, Wonnacott S. Differential inhibition by α-conotoxin-MII of the nicotinic stimulation of [3H] dopamine release from rat striatal synaptosomes and slices. J Neurochem 1998; 70: 1069-76 )。新近研究报道， 阻断含有 α3β4或 α6β2的 nAChRs可有效 防止烟瘾和吗啡毒瘾的发作， 显著抑制吸烟和吸毒的欲望 （Brunzell DH, Boschen KE, Hendrick ES, Beardsley PM, Mcintosh JM. Alpha-conotoxin Mil-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine. Neuropsychopharmacology,
2010;35(3):665-673. ) 。
此外， DA神经元中含有 α6亚基的 nAChRs表达量非常高， 由于 缺乏 α6* nAChRs 特异的药理学分子探针， α6 nAChR在成瘾中所具有 的重要作用的机制还不清楚。 哺乳动物脑中纹状体上的（¾6p2*-nAChRs 亚型被认为是治疗烟瘾和毒瘾的药物作用靶点 (Exley, R.; Clements, M. A.; Hartung, Η·; Mcintosh, J. Μ·; Cragg, S. J., Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens. Neuropsychopharmacology 2008,33 (9)， 2158-66)。 6亚^ ^脑中并不是 广泛分布的， 但却在中脑部分的多巴胺能神经元区域富集表达， 这个区 域是与愉快、 奖赏与心情控制密切相关的区域， 这意味着 a6* nAChRs 在药物引起的成瘾与情绪控制等的调控中起关键作用 （Yang, K. C.， G. Z. Jin, et al. (2009). Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology. Acta Pharmacol Sin 30(6): 740-751. Klink, R.; de Kerchove d'Exaerde, A.; Zoli, Μ·; Changeux, J. P., Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. The Journal of neuroscience, 2001,21 (5), 1452-63. Azam, L.; Winzer-Serhan, U. H.; Chen, Y.; Leslie, F. M.， Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. The Journal of comparative neurology 2002,444 (3), 260-74. Champtiaux, N.; Gotti, C; Cordero-Erausquin, Μ·; David, D. J.; Przybylski, C; Lena, C; Clementi, F.; Moretti, Μ·; Rossi, F. Μ·; Le Novere, N.; Mcintosh, J. Μ·; Gardier, A. Μ·; Changeux, J. P., Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. The Journal of neuroscience, 2003,23 (21), 7820-9. Pons, S.; Fattore, L.; Cossu, G.; Tolu, S.; Porcu, E.; Mcintosh, J. Μ·; Changeux, J. P.; Maskos, U.; Fratta, W.， Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration. The Journal of neuroscience, 2008,28 (47)， 12318-27)。 a6* nAChRs也表 i½儿茶臉胺 能神经元与视网膜上 (Le Novere, N.; Zoli, Μ·; Changeux, J. P., Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. The European journal of neuroscience 1996,8 (11), 2428-39. Vailati, S.; Hanke, W.; Bejan, A.; Barabino, Β·; Longhi, R.; Balestra, Β·; Moretti, Μ·; Clementi, F.; Gotti, C" Functional alpha6-containing nicotinic receptors are present in chick retina. Molecular pharmacology 1999,56 (1), 11-9.). α6β2* nAChRs 表现出具有调控多巴胺释放的功能， 在灵长类的 1 -methyl-4-phenyl- 1 ,2,3,6-tetr ahydropyridine动物模型与人类帕金森症 疾病模型上， α6β2* nAChRs的数量明显降低 (Champtiaux, N.; Han, Z. Υ·; Bessis, A.; Rossi, F. Μ·; Zoli, Μ·; Marubio, L.; Mcintosh, J. Μ·; Changeux, J. P., Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice. The Journal of neuroscience, 2002,22 (4), 1208-17. Quik, Μ·; Polonskaya, Y.; Kulak, J. Μ·; Mcintosh, J. M.， Vulnerability of 1251-alpha-conotoxin Mil binding sites to nigrostriatal damage in monkey. The Journal of neuroscience, 2001,21 (15), 5494-500. Quik, Μ·; Bordia, T.; Forno, L.; Mcintosh, J. M.， Loss of alpha-conotoxin Mil- and A85380-sensitive nicotinic receptors in Parkinson's disease striatum. Journal of neurochemistry 2004,88 (3), 668-79). 因而， «6/α3β2β3 nAChRs 的特异阻断剂是研究解释不同組织中存在的 α6 * nAChRs 生理功能的有价值的工具， 以及与之有关的如成瘾、 帕金森症 等疾病的治疗药物或药物筛选的工具药。
新近研究表明， 阻断含有 α3β4的 nAChRs可有效防止烟瘾、 吗啡 和可卡因毒瘾的发作， 显著抑制吸烟和吸毒的欲望 （Brunzdl DH, Boschen KE, Hendrick ES, Beardsley PM， Mcintosh JM. Alpha-conotoxin Mil-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine, Neuropsych oph armacology, 2010, 35(3):665-73 ) 。
据调查， 疼痛影响 1/6左右的人群， 包括关节炎、 神经痛、 肿痛。 其中神经痛影响 4 - 8 %的人群， 包括酒精中毒、 坐骨神经痛、 癌症与 癌症化疗、 糖尿病、 三叉神经痛、 硬化症、 带状疱疹、 机械伤和手术 伤等都会引起神经痛。 含有（¾3-亚基的 nAChRs, 包括 α3β2与 α3β4亚 型主要表达在外周神经系统， 中枢神经系统也有分布， 是神经痛药物作 用的靶点。阻断 α3β2或 α3β4 nAChRs的 α-芋螺毒素在临床前多种顽固 性疼痛 ( chronic pain )模型上显示出很好的镇痛活性， 且不成瘾。 顽 固性疼痛是一H¾：界性的健康难题， 急需新的治疗药物面世 ( apier, I. A.; Klimis, Η·; Rycroft, Β. Κ·; Jin, Α. Η·; Alewood, P. F.; Motin, L.; Adams, D. J.; Christie, M. J., Intrathecal α-conotoxins Vcl.l, AuIB and Mil acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain. Neuropharmacology 2012,62 (7), 2202-2207. Blyth, F. Μ·; March, L. Μ·; Brnabic, A. J.; Jorm, L. R.; Williamson, Μ·; Cousins, M. J., Chronic pain in Australia: a prevalence study. PAIN 2001,89 (2-3). 127-34. Cousins, M. J.; Brennan, F.; Carr, D. B.， Pain relief: a universal human right. PAIN 2004,772 (1-2), 1-4. Eisenberg, E.; McNicol, E. D.; Carr, D. B.， Efficacy and safety of opioid agonists in the treatment of neuropathic pain of nonmalignant origin: systematic review and meta-analysis of randomized controlled trials. JAMA： the journal of the American Medical Association 2005,293 (24), 3043-52. ) 。
α3β4 nAChRs是感觉的以及自律的神经中枢中主要的乙酰胆碱受 体亚型。 α3β4 nAChRs也是中枢神经系统（ CNS )神经元的分支， 譬如 向中枢延伸的松果体缰、 背部骨髓， 涉及到烟碱及其它滥用药物的成瘾 ( Millar, N. S.; Gotti, C" Diversity of vertebrate nicotinic acetylcholine receptors. Neuropharmacology 2009,56 (1), 237-46; Tapper, A. R.; McKinney, S. L.; Nashmi, R.; Schwarz, J.; Deshpande, P.; Labarca, C; Whiteaker, P.; Marks, M. J.; Collins, A. C; Lester, H. A., Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science 2004,306 (5698), 1029-32. )。 α3β4 nAChR 涉及到 中枢边缘多巴胺途径， 对于滥用某种物质（如毒品）产生的奖赏效应起 着非常重要的作用。 并且在 P4亚基敲除的老鼠中， 烟碱引起的运动和 奖赏效应显著减少， 这意味着（¾3p4 nAChR在 CNS 中烟碱成瘾的重要 作用 （ Salas, R" Sturm, R.， Boulter, J., and De Biasi, M. (2009) Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice. J. Neurosci. 29, 3014-3018 )。 α3β4 nAChRs 在恐惧反应中也起着很重要的作用，对于调节谷氛酸和去甲腎上腺素的 释放至关重要（ Zhu, P. J.; Stewart, R. R.; Mcintosh, J. M.; Weight, F. F., Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons. Journal of neurophysiology 2005,94 (5), 3081-91. Alkondon, Μ·; Albuquerque, E. X.， A non-alpha7 nicotinic acetylcholine receptor modulates excitatory input to hippocampal CAl interneurons. Journal of neurophysiology 2002,87 (3), 1651-4. Luo, S.; Kulak, J. Μ·; C artier, G. E.; Jacobsen, R. B.; Yoshikami, D.; Olivera, B. Μ·; Mcintosh, J. M.， alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. The Journal of neuroscience ： the official journal of the Society for Neuroscience 1998,18 (21)， 8571-9. Kulak, J. Μ·; Mcintosh, J. Μ·; Yoshikami, D.; Olivera, B. M.， Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels. Journal of neurochemistry 2001,77 (6), 1581-9. ) 。
对 nAChRs特定亚型具有非凡选择性的 α-CTxs是研究各种亚型分布 和功能的绝对必需的工具和相关疾病的治疗药物 （Kasheverov, I. E.， Utkin, Y. Ν., and Tsetlin, V. I. (2009) Naturally Occurring and Synthetic Peptides Acting on Nicotinic Acetylcholine Receptors. Current Pharmaceutical Design 15, 2430-2452; Nicke, A., Wonnacott, S.， and Lewis, R. J. (2004) alpha-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. Eur. J. Biochem. 271, 2305-2319 ) 。 特异阻断 α3β2 亚型， 而 对其极其相似的 α6β2* 亚型阻断活性很小或没有的 α-CTx具有非常珍 贵的价值， 也就是说能将 α3β2与 α6β2*两种亚型区分开的配体具有极其 重要的科学与应用价值。 因为， 在多巴胺能区域， 以 α6β2*亚型占主导 地位。 我们对这个重要生理功能区域的受体群的組成、 特征、 和生理作 用的了解仅仅来自 α-CTx ΜΠ的应用， 但是 α-CTx Mil对 (¾3β2与 (¾6β2*两种亚型的 选择性很差， 无法区分它们。 或者是用选择性的 α6β2*亚型的阻断剂来 研究 ( Dowell, C" Olivera, Β. Μ·， Garrett, J. Ε·， Staheli, S. Τ·， Watkins, Μ·， Kuryatov, A., Yoshikami, D., Lindstrom, J. M.， and Mcintosh, J. M. (2003) a-Conotoxin PIA Is Selective for a6 Subunit-Containing Nicotinic Acetylcholine Receptors. The Journal of Neuroscience 23, 8445-8452; Mcintosh, J. M.， Azam, L., Staheli, S.， Dowell, C.， Lindstrom, J. M.， Kuryatov, A., Garrett, J. E.， Marks, M. J., and Whiteaker, P. (2004) Analogs of alpha-conotoxin Mil are selective for alpha 6-containing nicotinic acetylcholine receptors. Molecular pharmacology 65, 944-952; Quik, M.， Perez, X. A., and Grady, S. R. (2011) Role of alpha 6 nicotinic receptors in CNS dopaminergic function: relevance to addiction and neurological disorders. Biochemical pharmacology 82, 873-882； Letchworth, S. R.， and Whiteaker, P. (2011) Progress and challenges in the study of alpha 6-containing nicotinic acetylcholine receptors. Biochemical pharmacology 82, 862-872； Champtiaux, N., Gotti, C.， Cordero-Erausquin, M.， David, D. J., Przybylski, C.， Lena, C.， Clementi, F., Moretti, M.， Rossi, F. M.， Le Novere, N., Mcintosh, J. M.， Gardier, A. M.， and Changeux, J. P. (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. Journal of Neuroscience 23, 7820-7829 )。然而, α3β2* nAChRs的表达往往会与 α6β2* nAChRs的表 ¾f互重叠， 有时 α3β2* nAChRs还占主导（ Whiteaker, P., Mcintosh, J. Μ·， Luo, S. Q., Collins, A. C" and Marks, M. J. (2000) I-125-alpha-conotoxin Mil identifies a novel nicotinic acetylcholine receptor population in mouse brain. Molecular pharmacology 57, 913-925； Whiteaker, P., Peterson, C. G., Xu, W., Mcintosh, J. M.， Paylor, R.， Beaudet, A. L., Collins, A. C.， and Marks, M. J. (2002) Involvement of the alpha 3 subunit in central nicotinic binding populations. Journal of Neuroscience 22, 2522-2529； McClure-Begley, T. D., Wageman, C. R" Grady, S. R.， Marks, M. J., Mcintosh, J. M.， Collins, A. C" and Whiteaker, P. (2012) A novel alpha-conotoxin Mil-sensitive nicotinic acetylcholine receptor modulates H-3 -GABA release in the superficial layers of the mouse superior colliculus. J Neurochem 122, 48-57 ) 。 此夕卜， 脊柱中的 α3β2* nAChRs在疼痛刺激的传递中起重要作用， 是镇痛药物作用的靶点 ( Young, Τ·， Wittenauer, S.， Mcintosh, J. M.， and Vincler, M. (2008) Spinal α3β2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli. Brain research 1229, 118-124 )一个真正的 α3β2* vs. α6β2* nAChRs选择性阻断剂的发现， 对 于综合研究理解该亚型在正常和疾病状态下的功能和意义具有非常重 要的价值。
可见， α-芋螺毒素在疼痛， 戒烟， 戒毒， 治疗帕金森病、 痴呆、 抑 郁和精神分裂等新药的研发、 以及相关疾病机理的研究等领域具有巨大 的潜力， 市场亦急需能区分特定 nAChRs亚型的分子探针工具药、 以及 治疗神经痛、 以及成瘾等的创新药物上市， 以緩解疼痛、 吸烟吸毒等带 来的疾病损失和严重的社会问题。 目前， 亟需发现新的高特异性的 nAChRs阻断剂。 发明内容 本发明人经过深入的研究和创造性的劳动， 发现了新的 α-芋螺毒 素肽， 其能够特异性地阻断乙酰胆碱受体， 特别是对神经痛药物靶点 α3β2 nAChRs, α3β4 nAChRs或 α6/α3β4 nAChRs, 以及成瘾药物乾点 «6/α3β2β3 nAChRs或 α3β4 nAChRs具有强阻断活性，并且在动物模型 上显示出极强的镇痛活性， 在制备镇痛、 戒烟戒毒药物， 防治抑郁、 痴呆、 精神分裂症、 帕金森症等疾病或作为神经科学工具药等方面具 有的良好应用前景。 由此提供了下述发明： 本发明的一个方面涉及一种多肽， 其具有如下的式 I所示的氛基 酸序列：
GCCSX XaCX X PX CX 式 I
其中，
表示 D或 H，
X2表示 P、 A或 V，
X3表示 R、 N或 S，
X4表示 N、 V或 A,
X5表示 K、 D、 M或 A，
X6表示 H或 S，
X7表示 D、 E或者 X7缺失，
X8表示 L或 I，
X9表示 G或者 X9缺失；
可选地， 式 I多肽的 C-末端酰胺化。
上述 ^酸0、 H、 P、 A、 V、 R、 N、 S、 K、 M、 H、 E、 L、 I、 G等是 ^酸的缩写， 其具有本领域人员公知的含义。
式 I 多 肽的 C-末端酰胺化也可以用 #表示， 即：
Figure imgf000013_0001
本发明还涉及一种多肽， 其为或者包含选自如下 （1 )至（3 ) 中 任一项所述的氛基酸序列： ( 1 ) SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 6、 SEQ ID NO: 11— 15、 SEQ ID NO: 26— 28或 SEQ ID NO: 30中的任一序列 所示的處基酸序列；
( 2 )与上述（ 1 )所述氛基酸序列至少 80%、 优选至少 85%、 更 优选至少 90%、 尤其优选至少 95%、 最优选至少 97%相同的氛基酸 序列； 或
(3)被 1-5个、 优选 1-3个、 更优选 1-2个、 最优选 1个氨 基酸残基的取代、 缺失、 插入和 /或添加而与上述（1)或（2)所示序 列有所不同的處基酸序列。
为了本发明的一个目的， 两个或更多个氛基酸序列之间的相同程 度是通过 BLAST2.0蛋白质数据库查询程序（ Aaltschul等， 1997， 核 酸研究 25: 3389 - 3402 )并采用下列参数确定的： blastall -p blastp-a4- el0-E0-v500-b250-I [查询文档】 -d prot all, 其中 -p指程序名称， -a指 将要用到的服务器数， -e指期望值， -E指延伸缺口的代价， -V指单线 描述（one-line description )数， -b指将要显示的比对数， -I指查询文 档， -d指用于查询的数据库。
同源多肽的 ^酸序列与 SEQIDNO: 3、 SEQ ID NO: 4、 SEQ ID NO: 6、 SEQ ID NO: 11 - 15、 SEQ ID NO: 26 - 28或 SEQ ID NO: 30中任一氛基酸序列不同之处可能在于取代、插入、添加和 /或缺失了 1或多个、 优选 1-5个、 更优选 1-3个、 尤其优选 1-2个、 最优选 1 个氛基酸残基。 优选地， 氛基酸改变是性质改变较小的变化， 即是 不会显著影响蛋白质的折叠和 /或活性的保守性 ^酸取代； 小片段缺 失， 通常是 1到大约 5个、 优选 1 - 3个、 更优选 1个氛基酸的缺失； 小的氛基或羧基末端延伸， 如氛基端添加的甲硫氨酸残基； 有多达大 约 20 - 25个残基的小连接肽；或可通过改变净电荷或者其它功能而有 助于纯化的小延伸如多聚組氨酸片段、 抗原表位或结合区。
保守性取代的例子是在碱性氛基酸（精氨酸、赖氨酸和組氨酸）、 酸性氛基酸（谷氨酸和天冬氨酸） 、 极性氛基酸（谷氨酰胺和天冬酰 胺）、 疏水氛基酸（亮氨酸、 异亮氨酸和缬氨酸）、 芳香族氛基酸（苯 丙氨酸、 色氨酸和酪氛酸）和小氛基酸（甘氛酸、 丙氨酸、 丝氨酸、 苏氨酸和甲硫氨酸） 内进行的取代。 通常不会改变特异活性的氛基酸 取代是本领域已知的， 并且由例如 H.Neurath和 R丄. Hill, 1979， 在 《蛋白质》一书， Academic Press, New York中描述过。 最常见的替 换是 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和 Asp/Gly以及反向进行的替换。
本发明还包括在本发明 α-芋螺毒素肽的 N-末端和 /或 C-末端融合 了其它肽 /多肽的融合多肽或可裂解的融合多肽。产生融合多肽的技术 为本领域内已知， 包括连接编码本发明肽的编码序列与编码所述其它 肽 /多肽的编码序列， 使它们在同一读框中， 并且融合多肽的表达受控 于相同的启动子和终止子。
根据本发明任一项所述的多肽， 其具有 SEQ ID NO : 4 ( α- LvIA/LvD21 ) 、 或 SEQ ID NO: 3 (该多肽实际上是 a- LvIA/LvD21 的前肽）所示的氛基酸序列。
根据本发明任一项所述的多肽， 其中， 所述多肽的 C末端是酰胺 化的。 所述酰胺化可以通过人工化学合成实现， 也可以在细胞内或细 胞外， 通过酰胺化酶实现。
根据本发明任一项所述的多肽， 其中， 所述多肽的 N末端的第一 个半胱氨酸与第三个半胱氨酸形成二硫键， 并且第二个半胱氨酸与第 四个半胱氨酸形成二硫键;或所述多肽的 N末端的第一个半胱氛酸与 第四个半胱氨酸形成二硫键， 并且第二个半胱氨酸与第三个半胱氨酸 形成二硫键； 所述多肽的 N末端的第一个半胱氨酸与第二个半胱氨酸 形成二硫键， 并且第三个半胱氛酸与第四个半胱氨酸形成二硫键。
本发明的上述多肽即为芋螺毒素肽； 具体地， 是 α-芋螺毒素肽。 上述芋螺毒素肽可以从我国海南产的疣缟芋螺（Conus lividus ) 或织锦芋螺 (C l"S ^ / )中提取。 也可以化学合成氛基酸序列 （例如 参考实施例 2-(1)至 2-(3)中的方法）；或者通过基因重組的手段表达其 核苷酸 (核苷酸序列的制备参考实施例 1-(1)至 1-(3)或者直接按照实施 例 2-(1)至 2-(3)中的方法进行多肽的人工合成）， 得到多肽。 也可以参 考下面的方法：
本发明的另一方面涉及本发明任一项所述的多肽的制备方法， 包 括下述步骤：
1 )在 ABI Prism 433a 多肽合成仪上或者手工方法合成线性多肽， Fmoc ^酸的侧链保护基为： Pmc(Arg)、 Trt或 Acm(Cys)、 But (Thr、 Ser、 Tyr)、 OBut (Asp)以及 Boc (Lys);
2)将步骤 1) 中合成的线性多肽从树脂上切割下来；
3)用冰乙醚沉淀和洗涤步骤 2) 中得到的线性多肽， 回收得到线 性多肽粗品；
4)用制备型反向 HPLC C18 柱（Vydac) 纯化步骤 3 ) 中得到的 线性多肽粗品；
5)将步骤 4) 中得到的产物进行两步或一步氧化折叠。 本发明的再一方面涉及一种多核苷酸， 其编码本发明任一项所述 多肽的處基酸序列。
根据本发明任一项所述的多核苷酸，其为或者包含选自如下的（ 1 ) 至（3) 中任一项所述的核苷酸序列：
( 1 ) SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 5、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 16-21、 SEQ ID NO: 22- 25、 SEQ ID NO: 29或 SEQ ID NO: 31中的任一序列所示的核苷酸 序列；
(2)上面 （1) 中所述核苷酸序列的互补序列；
(3)在严谊条件下能够与上述（1) 中所述的核苷酸序列杂交的 核苷酸序列。 关于多核苷酸之间的杂交，在现有技术中有众多的文献可供参考， 包括例如 Sambrook等， 分子克隆实验室手册， 第二版， 冷泉港实验 室， 冷泉港， 1989。 杂交中可以应用各种程度的严谊条件， 例如中度、 中度-高度， 或者高度严谊条件。 越严谊的条件， 形成双螺旋要求的 互补程度越高。 可以通过温度、 探针浓度、 探针长度、 离子强度、 时 间等等控制严谊程度。 对于默链 DNA基因探针， 杂交于低于 DNA杂 合体熔解温度 [melting temperature, Tm】)20 - 25。C下在 6X SSPE、 5X Denhardt氏溶液、 0.1 % SDS、 0.1mg/ml变性 DNA中进行过夜。 清洗 通常如下进行：于 Tm-20。C在 0.2X SSPE、0.1 % SDS中一次 15分钟（中 度严谊条件清洗)。 本发明的再一方面涉及一种核酸构建体， 其包含本发明任一项所 述的多核苷酸。
本发明的再一方面涉及一种表达载体， 其包含本发明任一项所述 的核酸构建体。
本发明的再一方面涉及一种转化的细胞， 其包含本发明任一项所 述的表达载体。
本发明的再一方面涉及一种融合蛋白， 其包含本发明任一项所述 的多肽。 本发明的再一方面涉及一种药物組合物， 其包含本发明任一项所 述的多肽， 或者包含本发明的融合蛋白； 可选地， 其还包含药学上可 接受的载体或辅料。 本发明的再一方面涉及一种阻断乙酰胆碱受体的方法， 包括使用 有效量的本发明任一项所述的多肽或本发明的融合蛋白的步骤； 具体 地， 所述乙酰胆碱受体是 α3β2 乙酰胆碱受体、 （X 6/ 0C 3 P 2 P 3乙酰胆 碱受体或 (¾3β4 乙酰胆碱受体。
本发明的再一方面涉及一种筛选乙酰胆碱受体抑制剂或者确定乙 酰胆碱受体亚型的方法， 该方法包括： 通过在存在或不存在候选化合 物存在的情况下将乙酰胆碱受体与本发明任一项所述的多肽或本发明 的融合蛋白进行接触的步骤； 具体地， 所述乙酰胆碱受体为 α3β2 乙 酰胆碱受体、 016/0130203乙酰胆碱受体或(¾3卩4 乙酰胆碱受体。 当 多肽或融合蛋白（例如 α-芋螺毒素 LvIA/LvD21 )能够特异阻断 α3β2 乙 酰胆碱受体、 （例如 α-芋螺毒素 TxIB/Txd4 )能够特异阻断 (X 6/ (X 3 P 2 P 3乙酰胆碱受体或 （例如 α-芋螺毒素 TxIC/Txdl) 能够特异阻断 α3β4 乙酰胆碱受体时， 则推断该乙酰胆碱受体分别是 α3β2亚型、 （Χ6Ρ2* 亚型 （（X6/0C3P2P3乙酰胆碱受体）或 α3β4亚型的乙酰胆碱受体。 本发明的再一方面涉及本发明任一项所述的多肽或本发明的融合 蛋白用于阻断乙酰胆碱受体的用途； 具体地， 所述乙酰胆碱受体是 α3β2乙酰胆碱受体、 （X6/0C3P2P3乙酰胆碱受体或 α3β4 乙酰胆碱受 体。
本发明的再一方面涉及本发明任一项所述的多肽或本发明的融合 蛋白在制备阻断乙酰胆碱受体的药物或试剂中的用途； 具体地， 所述 乙酰胆碱受体是 α3β2 乙酰胆碱受体、 （X6/0C3P2P3乙酰胆碱受体或 α3β4 乙酰胆碱受体。
本发明的再一方面涉及本发明任一项所述的多肽或本发明的融合 蛋白在制备治疗和 /或预防和 /或辅助治疗神经系统疾病例如成瘾、 神 经痛、 帕金森症、 或痴呆等的药物， 或者用于制备杀灭害虫、 镇痛、 戒烟、 或戒毒的药物的用途； 具体地， 所述神经痛由如下原因导致： 癌症与癌症化疗、 酒精中毒、 坐骨神经痛、 糖尿病、 三叉神经痛、 硬 化症、 带状疱疹、 机械伤和手术伤、 艾滋病、 头部神经瘫痪、 药物中 毒、 工业污染中毒、 淋巴神经痛、 骨髓瘤、 多点运动神经痛、 慢性先 天性感觉神经病、 急性剧烈自发性神经痛、 挤压神经痛、 脉管炎、 血 管炎、 局部缺血、 尿毒症、 儿童胆汁肝脏疾病、 慢性呼吸障碍、 复合 神经痛、 多器官衰竭、 脓毒病 /脓血症、 肝炎、 卟啉症、 维生素缺乏、 慢性肝脏病、 原生胆汁硬化、 高血脂症、 麻疯病、 莱姆关节炎、 感觉 神经束膜炎、 过敏症等。 本发明的再一方面涉及一种治疗和 /或预防和 /或辅助治疗神经系 统疾病例如疼痛、 烟酒和毒品成瘾、 智障、 痴呆、 精神分裂症、 中枢 神经紊乱、 癫痫症、 帕金森病、 精神病、 神经肌肉阻滞、 重症肌无力、 抑郁症、 高血压、 心率不齐、 哮喘、 肌肉松弛、 中风、 乳腺癌和肺癌等 的方法， 或者一种杀灭害虫、 镇痛、 戒烟、 或戒毒的方法， 包括给予 有效量的本发明的多肽（芋螺毒素肽或其前肽）或者本发明的药物組 合物或者本发明的融合蛋白的步骤； 具体地， 所述成瘾由烟碱、 吗啡、 可卡因、 酒精等能引起上瘾的物质； 所述神经痛由如下原因导致： 癌 症与癌症化疗、 酒精中毒、 坐骨神经痛、 糖尿病、 三叉神经痛、 硬化 症、 带状疱疹、 机械伤和手术伤、 艾滋病、 头部神经瘫痪、 药物中毒、 工业污染中毒、 淋巴神经痛、 骨髓瘤、 多点运动神经痛、 慢性先天性 感觉神经病、 急性剧烈自发性神经痛、 挤压神经痛、 脉管炎、血管炎、 局部缺血、 尿毒症、 儿童胆汁肝脏疾病、 慢性呼吸障碍、 复合神经痛、 多器官衰竭、 脓毒病 /脓血症、 肝炎、 卟啉症、 维生素缺乏、 慢性肝脏 病、 原生胆汁硬化、 高血脂症、 麻疯病、 莱姆关节炎、 感觉神经束膜 炎、 过敏症等。
本发明的芋螺毒素肽可通过结合 α3β2乙酰胆碱受体 (nAChR) 、 6/ 3 β 2 β 3乙酰胆碱受体或 α3β4 乙酰胆碱受体发挥作用，具有戒 除成瘾与镇痛的活性。 可应用于研究、 诊断和治疗成瘾、 神经痛、 帕 金森病、 痴呆、 精神分裂症、 抑郁等神经系统疾病、 以及作为有用的 分子探针用于研究等方面。 不同的 a-CTx对脊推动物受体的亲和性不 同， 有时相差几个数量级。 这种种系间的差异使得 a-CTx可作为有用 的探针用于研究脊推动物 nAChR的种系发生， 可作为分子探针来确 定 nAchRs的不同亚型。 它们是新药开发的候选药物、 先导药物和治 疗药物。 下面给出了本发明涉及的术语的解释。 神经痛
本发明所述多肽涉及到治疗各种神经痛的用途。 神经痛是周围或 中枢神经系统原发或继发性损害或功能障碍或短暂紊乱引起的疼痛， 表现为自发性疼痛、 痛觉超敏、 痛觉过敏等。 很多疾病都会引起神经 痛， 包括癌症与癌症化疗、 酒精中毒、 坐骨神经痛、 糖尿病、 三叉神 经痛、 硬化症、 带状疱疹、 机械伤和手术伤、 艾滋病、 头部神经瘫痪、 药物中毒、 工业污染中毒、 淋巴神经痛、 骨髓瘤、 多点运动神经痛、 慢性先天性感觉神经病、 急性剧烈自发性神经痛、 挤压神经痛、 脉管 炎（血管炎） /局部缺血、尿毒症、 儿童胆汁肝脏疾病、慢性呼吸障碍、 复合神经痛、 多器官衰竭、 脓毒病 /脓血症、 肝炎、 卟啉症、 维生素缺 乏、 慢性肝脏病、 原生胆汁硬化、 高血脂症、 麻疯病、 莱姆关节炎、 感觉神经束膜炎、 过敏症等。 成癃 ( addiction )
本发明所述多肽涉及到能治疗各种有依赖性物质引起的成瘾。 成 瘾是指反复使用精神活性物质者处于周期性或慢性中毒状态。 精神活 性物质指尼古丁、 鸦片、 海洛因、 甲基苯丙胺（冰毒）、 吗啡、 大麻、 可卡因以及国家规定管制的其它能够使人形成瘾癖的麻醉药品和精神 药品等。 成瘾与大脑中大量产生的多巴胺（Dopamine )有关。 表现为 不可遏制地应用偏爱的物质和难以自制或难以矫正使用行为， 为获取 精神活性物质达到感觉良好或避免戒断痛苦之目的， 可以不择手段。 典型情况是耐受性增高， 并在物质使用中断后常出现戒断症状。 成瘾 者的生活可能完全由物质使用主宰， 因而受到严重影响， 甚至抛弃了 其它重要活动和一切责任。 因此， 物质使用既给个人， 也给社会带来 损害。 当用于酒精使用时， 与慢性酒精中毒的概念等同。 成瘾一词还 涵盖躯体及心理两方面的内容。 心理成瘾强调对饮酒、 服药的自控力 受损体验， 而躯体成瘾指耐受和戒断症状。 核酸构建体
本发明还涉及包含本发明所述核酸序列及与之可操作连接的 1或 多个调控序列的核酸构建体， 所述调控序列在其相容条件下能指导编 码序列在合适的宿主细胞中进行表达。 表达应理解为包括多肽生产中 所涉及的任何步骤， 包括， 但不限于转录、 转录后修饰、 翻译、 翻译 后修饰和分泌。
"核酸构建体 "在文中定义为单链或默链核酸分子， 它们分离自天 然基因， 或者经修饰而含有以非天然方式組合和并列的核酸片段。 当 核酸构建体包含表达本发明所述编码序列必需的所有调控序列时， 术 语核酸构建体与表达盒同义。 术语"编码序列，，在文中定义为核酸序列 中直接确定其蛋白产物的 ^酸序列的部分。 编码序列的边界通常是 由紧邻 mRNA 5，端开放读码框上游的核糖体结合位点 (对于原核细胞) 和紧邻 mRNA 3，端开放读码框下游的转录终止序列确定。编码序列可 以包括， 但不限于 DNA、 cDNA和重組核酸序列。
可以以多种方式操作编码本发明所述肽的分离的核酸序列， 使其 表达所述肽。 可能期望或必须在插入载体之前对核酸序列进行加工， 这取决于表达载体。 应用重組 DNA方法修饰核酸序列的技术为本领 域所熟知。
本文中术语"调控序列，，定义为包括表达本发明肽所必需或有利的 所有組分。 每个调控序列对于编码多肽的核酸序列可以是天然含有的 或外来的。 这些调控序列包括， 但不限于， 前导序列、 多聚腺苷酸化 序列、 前肽序列、 启动子、 信号序列和转录终止子。 最低限度， 调控 序列要包括启动子以及转录和翻译的终止信号。 为了导入特定的限制 位点以便将调控序列与编码多肽的核酸序列的编码区进行连接， 可以 提供带接头的调控序列。 术语"可操作连接，，在文中定义为这样一种构 象， 其中调控序列位于相对 DNA序列之编码序列的适当位置， 以使 调控序列指导多肽的表达。
调控序列可以是合适的启动子序列， 即可被表达核酸序列的宿主 细胞识别的核酸序列。启动子序列含有介导多肽表达的转录调控序列。 启动子可以是在所选宿主细胞中有转录活性的任何核酸序列， 包括突 变的、 截短的和杂合的启动子， 可以得自编码与宿主细胞同源或异源 的胞外或胞内多肽的基因。
调控序列还可以是合适的转录终止序列， 即能被宿主细胞识别从 而终止转录的一段序列。 终止序列可操作连接在编码多肽的核酸序列 的 3，末端。 在所选宿主细胞中可发挥功能的任何终止子都可以用于本 发明。
调控序列还可以是合适的前导序列， 即对宿主细胞的翻译十分重 要的 mRNA非翻译区。前导序列可操作连接于编码多肽的核酸序列的 5，末端。 在所选宿主细胞中可发挥功能的任何前导序列均可用于本发 明。
调控序列还可以是信号肽编码区， 该区编码一段连在多肽氛基端 的氛基酸序列， 能引导编码多肽进入细胞分泌途径。 核酸序列编码区 的 5，端可能天然含有翻译读框一致地与分泌多肽的编码区片段自然连 接的信号肽编码区。 或者， 编码区的 5，端可含有对编码序列是外来的 信号肽编码区。 当编码序列在正常情况下不含有信号肽编码区时， 可 能需要添加外来信号肽编码区。 或者， 可以用外来的信号肽编码区简 单地替换天然的信号肽编码区以增强多肽分泌。 但是， 任何能引导表 本发明。
调控序列还可以是肽原编码区， 该区编码位于多肽氛基末端的一 段氛基酸序列。所得多肽被称为酶原或多肽原。多肽原通常没有活性， 可以通过催化或自我催化而从多肽原切割肽原而转化为成熟的活性多 肽。
在多肽的氛基末端即有信号肽又有肽原区时， 肽原区紧邻多肽的 末端， 而信号肽区则紧邻肽原区的氛基末端。
添加能根据宿主细胞的生长情况来调节多肽表达的调控序列可能 也是需要的。 调控系统的例子是那些能对化学或物理刺激物 （包括在 有调控化合物的情况下）作出反应，从而开放或关闭基因表达的系统。 调控序列的其它例子是那些能使基因扩增的调控序列。在这些例子中， 应将编码多肽的核酸序列与调控序列可操作连接在一起。
表达载体
本发明还涉及包含本发明核酸序列、 启动子和转录及翻译终止信 号的重組表达载体。 可以将上述各种核酸和调控序列连接在一起来制 备重組表达载体， 该载体可以包括 1或多个方便的限制位点， 以便在 这些位点插入或取代编码多肽的核酸序列。 或者， 可以通过将核酸序 列或包含该序列的核酸构建体插入适当表达载体而表达本发明所述核 酸序列。 制备表达载体时， 可使编码序列位于载体中以便与适当的表 达调控序列可操作连接。
重組表达载体可以是任何便于进行重組 DNA操作并表达核酸序 列的载体（例如质粒或病毒） 。 载体的选择通常取决于载体与它将要 导入的宿主细胞的相容性。 载体可以是线性或闭环质粒。
载体可以是自主复制型载体（即存在于染色体外的完整结构， 可 独立于染色体进行复制） ， 例如质粒、 染色体外元件、 微小染色体或 人工染色体。 载体可包含保证自我复制的任何机制。 或者， 载体是一 个当导入宿主细胞时， 将整合到基因組中并与所整合到的染色体一起 复制的载体。 此外， 可应用单个载体或质粒， 或总体包含将导入宿主 细胞基因組的全部 DNA的两个或多个载体或质粒， 或转座子。
优选本发明所述载体含有 1 或多个便于选择转化细胞的选择标 记。选择标记是这样一个基因，其产物赋予对杀生物剂或病毒的抗性、 对重金属的抗性， 或赋予营养缺陷体原养型等。 细菌选择标记的例子 如枯草芽孢杆菌或地衣芽孢杆菌的 dal基因， 或者抗生素如氨苄青霉 素、 卡那霉素、 氯霉素或四环素的抗性标记。
优选本发明所述载体包含能使载体稳定整合到宿主细胞基因組 中， 或保证载体在细胞中独立于细胞基因組而进行自主复制的元件。
就进行自主复制的情况而言， 载体还可以包含复制起点， 使载体 能在目标宿主细胞中自主地复制。 复制起点可以带有使其在宿主细胞 中成为温度敏感型的突变 （参见例如， fEhrlich，1978， 美国国家科学 院学报 75: 1433 ) 。
可以向宿主细胞插入 1个以上拷贝的本发明核酸序列以提高该基 因产物的产量。 该核酸序列的拷贝数增加可以通过将该序列的至少 1 个附加拷贝插入宿主细胞基因組中， 或者与该核酸序列一起插入一个 可扩增的选择标记， 通过在有合适选择试剂存在下培养细胞， 挑选出 含有扩增拷贝的选择性标记基因、从而含有附加拷贝核酸序列的细胞。
用于连接上述各元件来构建本发明所述重組表达载体的操作是本 领域技术人员所熟知的（参见例如 Sambrook等， 分子克隆实验手册， 第二版， 冷泉港实验室出版社， 冷泉港， 纽约， 1989 ) 。 宿主细
本发明还涉及包含可用来重組生产多肽的本发明所述核酸序列的 重組宿主细胞。 可将包含本发明之核酸序列的载体导入宿主细胞， 从 而使该载体以上述染色体整合体或自我复制的染色体外载体形式得以 维持。 术语"宿主细胞，，涵盖任何由于复制期间发生的突变而与亲本细 胞不同的后代。 宿主细胞的选择很大程度上取决于多肽编码基因及其 来源。
宿主细胞可以是原核细胞或者真核细胞， 例如细菌或酵母细胞。 可以通过本领域技术人员熟知的技术将载体导入宿主细胞。 制备方法
本发明还涉及重組制备本发明肽的方法， 该方法包括： （a )在适 于产生所述肽的条件下， 培养含有核酸构建体的宿主细胞， 该核酸构 建体包含编码所述肽的核酸序列； 和 （b ) 回收该肽。
在本发明所述制备方法中， 用本领域已知方法在合适多肽产生的 营养培养基中培养细胞。 例如， 可以在合适的培养基中， 在允许多肽 表达和 /或分离的条件下， 通过摇瓶培养、 实验室或工业发酵罐中小规 模或大规模发酵（包括连续、 分批、 分批加料或固态发酵）来培养细 胞。 在包含碳和氮源以及无机盐的合适的培养基中， 采用本领域已知 的步骤进行培养。 合适的培养基可由供应商提供或者可以参照公开的 組成（例如， 美国典型培养物保藏中心的目录中所述）来制备。 如果 多肽被分泌到培养基中， 则可以直接从培养基中回收多肽。 如果多肽 不分泌， 可以从细胞裂解物中回收。
可以用本领域已知方法回收所产生的多肽。 例如， 可以通过常规 操作 （包括， 但不限于离心、 过滤、 抽提、 喷雾干燥、 蒸发或沉淀） 从培养基中回收多肽。
可以通过各种本领域已知的操作来纯化本发明所述多肽， 这些操 作包括， 但不限于层析（例如， 离子交换层析、 亲和层析、 疏水作用 层析、 层析聚焦、 和大小排阻层析） 、 HPLC、 电泳（例如， 制备性 等电点聚焦） 、 差示溶解度（例如硫酸铵沉淀） 、 SDS-PAGE或抽提 (参见例如， 蛋白质纯化， J.C.Janson 和 Lars Ryden 编， VCH Publishers, New York, 1989 ) 。 转基因动物和植物
本发明还涉及转化了本发明的核酸序列的动物或植物细胞， 优选 小麦、 玉米等植物细胞， 赋予被转化宿主新的性状（如抗虫性） 。 这 可以通过本领域技术人员熟知的技术， 用此处公开的构建体转化动物 或植物细胞而实现。 用于控制害虫的方法和制剂
可以通过本领域技术人员知道的多种方法， 使用本发明的芋螺毒 素肽或多核苷酸来实现控制害虫。 这些方法包括例如将重組微生物应 用于害虫 （或它们的所在地） 、 和用编码本发明的芋螺毒素肽的基因 转化植物。 转化可以由本领域技术人员使用常规技术进行。 此处公开 了用于这些转化的必要物质， 或者熟练的技术人员可以通过其它方法 容易的获得。
可以将配制的含有芋螺毒素肽、 或包含本发明所述多核苷酸的重 組微生物的制剂应用于土壤。 还可以将配制的产品作为种子覆料或根 部处理或作物生长周期晚期的完整植林处理应用。 制剂可以包括扩散 -增稠佐剂、 稳定剂、 其它杀虫添加剂、 或表面活性剂。 液体制剂可 以是基于水的或非水的， 并以泡沫、 凝胶、 悬浮液、 可乳化浓缩物等 等形式使用。 成分可以包括流变剂、 表面活性剂、 乳化剂、 分散剂、 或聚合物。
本领域技术人员可以理解， 杀虫剂浓度将由于特殊制剂的本性广 泛变化， 特别是可作为浓缩物或直接使用。 杀虫剂将以至少 1 % )重 量计）存在， 而且可能是 100 % (重量计） 。 干燥制剂通常有大约 1 - 95 % (重量计） 的杀虫剂， 而液体制剂将通常是液相中固体重量大 约 1 - 60 %。 含有细胞的制剂将通常含有大约 102 -大约 104个细胞 /mgo 这些制剂将以每公顷大约 50mg (液体的或干的） - 1kg或更多 的量使用。 通过喷、 撒、 洒等等， 可以将制剂应用于害虫环境， 例如 土壤和植物。 药物組合物
本发明还涉及含有本发明肽和药学可接受载体和 /或赋形剂的药 物組合物。 所述药物組合物可用于研究、 诊断、 緩解或治疗与成瘾、 神经痛、 智障、 疼痛、 帕金森症、 精神病、 抑郁、 重症肌无力、 癌症 等有关的疾病或病症。 在一个实施方案中， 含有治疗有效量的本发明 肽的药物組合物以利于药用的方式配制和给药， 并需考虑到个体病人 的临床状况、 运送位点、 给药方法、 给药日程安排和医生已知的其它 因素。 因此用于本文目的的 "有效量，，由这些方面的考虑决定。
含治疗有效量的本发明多肽的药物組合物非肠道给药、 口服、 脑 池内给药、 鞘内给药等。 "药学可接受载体"指无毒的固体、 半固体或 液体填充物、 稀释液、 胶嚢材料或任何类型的配方辅助物。 本文所用 术语"非肠道的"表示的给药方式包括静脉内、 肌肉内、 腹膜内、 胸骨 内、 皮下、 鞘内和关节内注射和输注。 本发明多肽还可通过緩幹系统 恰当地给药。
本发明还涉及特异阻断 nAChRs的药物組合物。
可应用本发明的芋螺毒素肽作为有用的探针来用于研究动物 nAChRs的种系发生； 作为分子探针来确定 nAChRs的不同亚型； 作 为分子模型，设计新药； 作为研究、 诊断神经性疾病如成瘾、 帕金森氏 病、 行动障碍、 精神分裂症等的工具药和治疗药物； 治疗乳腺癌、 肺 癌、 小细胞肺癌等的侯选药物。 作为多肽杀虫剂， 开发为新型生物农 药等。 发明的有益效果
本发明的 α-芋螺毒素肽能够特异地阻断乙酰胆碱受体 ( nAChRs ) , 并且具有强镇痛与戒除成瘾活性， 以及治疗帕金森症、 痴呆、 精神分裂症、 抑郁等疾病的功效。 附图说明
图 1: α-芋螺毒素 LvIA/LvD21 ( LvIA )前肽基因序列及其编码 产生的前肽与翻译后修饰产生的成熟肽。 箭头所指为翻译后修饰的加 工位点。推断的蛋白酶水解加工位点 1 ( processing 1 ) 在碱性氛基酸 精氨酸（R ) 的后面； C-末端酰胺化加工位点在箭头所指的甘氨酸的 位置， 用字符底纹表示， 即 processing 2. 成熟肽 C-末端紧挨半胱氛 酸（Cys ) 的第一甘氨酸残基往往是酰胺化翻译后修饰的加工位点， 从 processing site 2进行酰胺化产生的成熟肽命名为 LvIA/LvD21 (或 LvIA )，序列为： GCCSHPACNVDHPEIC# (#表示 C-末端酰胺化）。 前肽区用斜体字表示， 成熟肽用下划线表示， 其中的半胱氨酸（C ) 用黑体字显示， 终止密码子用 *表示。
图 2: 合成的线性肽与成熟肽 a-LvIA/LvD21 ( SEQ ID NO: 4 ) 序列及其二硫键连接方式 Ι-ΠΙ, II-IV, 及其相应的 HPLC 色谱图。 图 2A显示的是合成的线性肽序列， 及其 Cysl和 Cys3的自由 -SH与 Cys2和 Cys4上的保护基 S-Acm (S-acetamidomethyl); 图 2B显示的 是氧化折叠后的成熟肽 a-LvIA/LvD21序列， 及其含有的 I-III, II-IV 二硫键连接方式； 图 2C显示的是图 2A中合成的线性肽的 HPLC色 谱图， 其保留时间为 27.713min; 图 2D显示的是图 2B 中氧化肽的 HPLC色傳图， 其保留时间为 27.947min。
图 3: 图 3A显示的是 100 nM a-LvIA/LvD21 对 α3β2 nAChR的 电流影响情况， 钳制电压为 70 mV， 图 3A中的 "C"是指的对照电流， 箭头所指的是 100 nM a-LvIA/LvD21温育 5分钟后，第一个 Ach脉冲 形成的电流轨迹（ ~ 0 nA )，每两个电流轨迹之间的时间间隔为 l min。 图 3B、 3C、 3D分别是 a-LvIA/LvD21对各种 nAChRs亚型 （大鼠 11 个，人类 2个）的浓度剂量反应曲线，图中横坐标为所用 a-LvIA/LvD21 的摩尔浓度（M ) 的对数值 ( Log[LvIA/LvD21]M ) ；纵坐标为剂量反 应百分数（ % Response )， 是相应浓度的毒素作用下乙酰胆碱受体电 流与对照电流的比值百分数。 图 3B显示， a-LvIA/LvD21高选择性阻 断大鼠 α3β2 vs. «6/α3β2β3 nAChRs的浓度剂量反应曲线；图 3C显示， a-LvIA/LvD21对其他大鼠神经型亚型和小鼠肌肉型 nAChRs的浓度 剂量反应曲线；图 3D显示， a-LvIA/LvD21高选择性阻断人类 α3β2 vs. «6/α3β2β3 nAChRs的浓度剂量反应曲线。 图 3 中各个数值是取自 3 - 9个非洲爪蟾卵母细胞的电流平均值。
图 4: 不同剂量的 a-LvIA/LvD21各种 nAChRs的电流影响情况。 图 4A， 100 nM a-LvIA/LvD21 对大鼠 α3β2 nAChRs的电流影响情况； 图 4B, 10 μΜ a-LvIA/LvD21 对 α2β2 nAChRs的电流影响情况； 图 4C， 10 μΜ a-LvIA/LvD21 对小鼠肌肉型（Μαΐβδε) nAChRs 的电流 影响情况。 图 4 中 "C"指的是对照电流， 紧接" C，，后面的是 a-LvIA/LvD21的毒素浓度。箭头所指的是温育 5分钟后， LvIA/LvD21 阻断相应受体亚型的第一个 Ach脉冲形成的电流轨迹。图 4显示， 100 nM a-LvIA/LvD21 特异阻断 α3β2 nAChR, 而 10 μΜ 完全不阻断 α2β2(Β)与 Μα1βδε(< ) nAChRs亚型。
图 5: a-LvIA/LvD21对 α3β2 nAChRs及其 7个 β2突变型的浓度 剂量反应曲线。 图 5Α中的突变型是（¾3P2[F119Q】、 α3β2[νΐ11Ι] ; 图 5Β中的突变型是 (¾3P2[F119Q】、 α3β2[Τ59Κ] , «3p2[T59L] ; 图 5C中 的突变型是 α3β2[Τ59Ι】、 α3β2[Κ79Α] , a3p2[Q34A】。
图 6: 显示的是 10 nM a-LvIA/LvD21 对大鼠 α3β2 nAChRs野生 型（ A )，以及突变型 «3P2[F119Q] (Β)， α3β2[Τ59Κ] (C)与 α3β2[νΐ11Ι] (D)的电流影响情况， 以及阻断后不同的洗脱速率。 图 6A， 10 nM a-LvIA/LvD21阻断 α3β2 nAChRs野生型大约 50 %的电流， 洗脱速度 快， 2 min内电流完全恢复； 图 6B， 10 nM a-LvIA/LvD21阻断突变 型（¾3P2[F119Q】的全部电流，洗脱速度慢，洗脱 12 min后电流才恢复； 图 6C， 10 nM a-LvIA/LvD21阻断突变型 α3β2[Τ59Κ】的全部电流， 洗 脱速度非常慢， 洗脱 20 min后的电流才恢复至对照电流的 27 %。 图 6D, 10 nM a-LvIA/LvD21完全不阻断突变型 α3β2[νΐ11Ι】的电流。 图 中 "C"是指的对照电流， 箭头所指的是 10 nM a-LvIA/LvD21温育 5 分钟后， 第一个 Ach脉冲形成的电流轨迹 ( ~ 0 nA ) , "washout，，是 指洗脱， 每两个电流轨迹之间的时间间隔为 1 min。
图 7: a-LvIA/LvD21腹腔给药 （ IP )后 1 - 24h在 CCI模型上的 镇痛作用。 图中阴性对照 Saline为生理盐水 (Saline), 阳性对照为吗啡 ( Morphine ) ， 其给药剂量为 lmg/kg大鼠体重； a-LvIA/LvD21 的 给药剂量为 1 nmol/kg大鼠体重。图中横坐标 Time(hours)为给药后的 小时数； 纵坐标 Mechanical Threshold 为观测痛阈值与基础痛阈值
( 100 )的比值百分数 ( % of basal )， 图中各个点的纵坐标是平均值 和标准误（Mean±SD ) 。 显著性差异比较概率为 #p<0.05， 每組大鼠 数量为 8只 （ n=8 ) 。
图 8: a-LvIA/LvD21腹腔给药 （ IP )后 7-14天在 CCI模型上的 镇痛作用。 图中阴性对照 Saline为生理盐水 (Saline), 阳性对照为吗啡
( Morphine ) ， 其给药剂量为 lmg/kg大鼠体重； a-LvIA/LvD21 的 给药剂量为 1 nmol/kg大鼠体重。 图中横坐标 Time(days)为给药后的 天数；纵坐标 Mechanical Threshold为观测痛阈值与基础痛阈值（ 100 ) 的比值百分数 ( % of basal ) ， 图中各个点的纵坐标是平均值和标准 误（Mean±SD )。 显著性差异比较概率为 #ρ<0·05， 每組大鼠数量为 8 只 （ η=8 ) 。
图 9: a-LvIA/LvD21脑室给药 （ ICV )后 120 min内在小鼠热板 试验模型上的镇痛作用。 图中阴性对照 Saline为生理盐水 (Saline), 阳 性对照为吗啡 （Morphine ) ， 其给药剂量为 100 g/kg 小鼠体重； a-LvIA/LvD21 的给药剂量为 0.1 nmol/kg 小鼠体重。 图中横坐标 Time(min)为给药后的分钟数；纵坐标 Threshold ( sec )为观测痛阈值， 单位为秒。 图中各个点的纵坐标是平均值和标准误 ( MeaniSD ) 。 显 著性差异比较概率为 p<0.05， 每組小鼠数量为 10只 （n=10 ) 。
图 10: (X -芋螺毒素 TxIB/Txd4 ( TxIB )前肽基因序列及其编码 产生的前肽与翻译后修饰产生的成熟肽。 箭头所指为翻译后修饰的加 工位点。 推断的蛋白酶水解加工位点 1 ( processing site 1 ) 在碱性氛 基酸精氛酸（R ) 的后面； C-末端酰胺化加工位点可能在箭头所指的 两个甘氛酸的位置， 用字符底纹表示， 即 processing site 2 或 processing site 3. 成熟肽 C-末端紧挨半胱氛酸（ Cys ) 的第一或第二 个甘氨酸残基往往是酰胺化翻译后修饰的加工位点，从 processing site 2进行酰胺化产生的成熟肽命名为 TxIB/Txd4 (或 TxIB) ， 序列为： GCCSDPPCRNKHPDLC# (# 表示 C-末端酰胺化）； 从 processing site 3进行酰胺化产生的成熟肽命名为 TxIB/Txd4 (G) (或 TxIB(G) ) ， 序列为： GCCSDPPCRNKHPDLCG# (#表示 C-末端酰胺化）。 TxIB(G) 的 C-末端只比 TxIB多一个甘氛酸（ G )， 是 TxIB的类似物。 前肽区 用斜体字表示， 成熟肽用下划线表示， 其中的半胱氨酸（C)用黑体 字显示， 终止密码子用 *表示。
图 11:显示的是 (X-TxIB/Txd4(图 11A )以及 TxIB ( G ) (图 11B) 的成熟肽序列及其二硫键连接方式 I-III, II-IVo
图 12: (X -TxIB与 TxIB(G)是 (¾6/a3p2p3iiAChR的高选择性特异 阻断剂。图 12A显示的是 ΙμΜ a -TxIB 对（¾6/α3β2β3 nAChR的电流 影响情况。图 12A中" C"是指的对照电流，箭头所指的是 ΙμΜ a -TxIB 温育 5分钟后， 第一个 Ach脉冲形成的电流轨迹（ ~0nA) 。 图 12B 显示的是 (X -TxIB与 TxIB ( G)对（¾6/α3β2β3 nAChR的浓度剂量反 应曲线， 图中横坐标为所用 oc-TxIB与 TxIB(G)的摩尔浓度（M) 的 对数值 (Log [TxIB and TxIB(G)]M )；纵坐标为剂量反应百分数 (% Response ) ， 是相应浓度的毒素作用下乙酰胆碱受体电流与对照电流 的比值百分数。 图 12C显示的是 oc-TxIB对各种 nAChRs亚型的浓度 剂量反应曲线， （X -TxIB特异阻断 α6/α3β2β3 nAChR, 其半阻断剂量 ( IC50 )为 28 nM, 在 10 μΜ毒素浓度下， TxIB对其它亚型没有阻 断作用， 其 IC50 > 10 μΜ。 图中各个数值是取自 3-5个非洲爪蟾卵母 细胞的电流平均值。
图 13:显示的是 1 μΜ -ΤχΙΒ对（¾6/α3β2β3 nAChR (图 13A)， 以及 10 μΜ -ΤχΙΒ对其非常接近的（χ3β2 (图 13B)， α6/α3β4(图 13C), oc3P4(图 13D) nAChRs 的电流影响情况。 图中 "C"是指的对 照电流， 紧接 "C" 后面的是 (X -TxIB的毒素浓度。 箭头所指的是温育 5分钟后， TxIB阻断相应受体亚型的第一个 Ach脉冲形成的电流轨迹。 1 μΜ -ΤχΙΒ特异阻断（¾6/α3β2β3 nAChR (图 13A )， 而 10 μΜ完全 不阻断 (Χ 3 β 2 (图 13B )， （¾6/α3β4(图 13C)与（Χ 3 β 4(图 13D) nAChRs 亚型。
图 14: α-芋螺毒素 TxIC/Txdl ( TxIC )前肽基因序列及其编码产 生的前肽与翻译后修饰产生的成熟肽。 箭头所指为翻译后修饰的加工 位点。 推断的蛋白酶水解加工位点 1 ( processing site 1 ) 在碱性氛基 酸精氨酸（R ) 的后面； C 末端酰胺化加工位点在箭头所指的甘氛酸 的位置， 用字符底纹表示， 即 processing site 2. 成熟肽 C末端紧挨半 胱氨酸（Cys ) 的第一个甘氨酸残基往往是酰胺化翻译后修饰的加工 位点，从 processing site 2进行酰胺化产生的成熟肽命名为 TxIC/Txdl (或 TxIC ) ， 序列为： GCCSHPVCSAMSPIC # (# 表示 C末端酰胺 化)。前肽区用斜体字表示，成熟肽用下划线表示，其中的半胱氛酸（ C ) 用黑体字显示， 终止密码子用 *表示。
图 15: A显示的是成熟肽 a-TxIC/Txdl ( SEQ ID NO: 28 )序列 及其二疏键连接方式 Ι-ΠΙ, II-IV。 Β显示的是含有 Ι-ΠΙ, II-IV二疏 键连接方式的 α-TxIC/Txdl的 HPLC色谱图，该毒素肽的色谱分析条 件为： 用 Vydac C18 HPLC反相分析柱， 在 40分钟内进行线性梯度 洗脱， B液从 15%到 50%, A液从 85%到 50%, A液是 0.65% 的三 氟乙酸（ trifluoroacetic acid, TFA) , B 是 0.5% TFA 与 90%乙腈 ( acetonitrile ) 的水溶液。 紫外分析波长为 214nm， TxIC 的出峰时 间， 即保留时间是 23.366分钟。
图 16: α-TxIC «3β4 nAChR的选择性强阻断剂。 A显示的是 ΙμΜ α-TxIC 对 α3β4 nAChR的电流影响情况。 图 A中" C，，是指的对 照电流， 箭头所指的是 ΙμΜ α-TxIC温育 5分钟后， 第一个 Ach脉冲 形成的电流轨迹（ ~ 0 nA )。 B显示的是 α-TxIC对其它 10个 nAChRs 各种亚型的浓度剂量反应曲线， 图中横坐标为所用 α-TxIC 的摩尔浓 度 （M ) 的对数值 （Log[TxIC】M ) ；纵坐标为剂量反应百分数（％ Response ) ， 是相应浓度的毒素作用下乙酰胆碱受体电流与对照电流 的比值百分数。 α-TxIC特异阻断 (¾3p4 iiAChR， 其半阻断剂量（IC50 ) 仅为 12.5 nM; α-TxIC对 α6/α3β4 nAChR也有一定的阻断作用，其半 阻断剂量（ IC5。 )为 94 nM; α-TxIC对 α2β4 nAChR有很微弱的阻断 作用， 其半阻断剂量高达 4550 nM。 在 10 μΜ毒素浓度下， TxIC对 其它亚型没有阻断作用， 其 IC5。> 10 M。 图中各个数值是取自 3 - 8 个非洲爪蟾卵母细胞的电流平均值。
图 17:显示的是 1 μΜ α-TxIC对 α3β4 nAChR ( A )， 以及 10 μΜ α-TxIC对其 接近的 α4β4(Β)， a7(C) nAChRs 的电流影响情况。 图 中" C"是指的对照电流， 紧接" C"后面的是 a-TxIC的毒素浓度。 箭头 所指的是温育 5分钟后， TxIC阻断相应受体亚型的第一个 Ach脉冲形 成的电流轨迹。 1 μΜ α-TxIC特异阻断 α3β4 nAChR, 而 10 μΜ完全 不阻断 α4β4(Β)与（¾7(C) nAChRs亚型。 具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。 本领域技 术人员将会理解， 下面的实施例仅用于说明本发明， 而不应视为限定 本发明的范围。 实施例中未注明具体技术或条件者， 按照本领域内的 文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译 的《分子克隆实验指南》， 第三版， 科学出版社）、 相应的参考文献、 或者按照产品说明书进行。 所用试剂或仪器未注明生产厂商者， 均为 可以通过市购获得的常规产品。 实施例 1-α): α-芋螺毒素 LvIA/LvD21基因的克隆和序列分析 1. 疣缟芋螺基因組 DNA的提取
分别以从海南岛、 西沙群岛等沿海采集的疣缟芋螺 (C. textile Linnaeus)活体为材料， 储存在 -80。C备用。 先将芋螺毒腺解剖出来， 并称重。 然后用海洋动物基因組 DNA提取试剂盒（购自中国北京天 根生化科技有限公司）， 提取毒腺的基因組 DNA, 具体操作参见试剂 盒说明书； 也可以参考文献， 郑晓冬、 高炳淼、 李宝珠、 彭超、 吴爱 银、 朱晓鹏、 陈心、 长孙东亭、 罗素兰， 新型 α-芋螺毒素基因克隆的 引物筛选， 生物技术， 2011， 21 ( 4 ) ： 40-44。
将提取的毒腺基因組总 DNA溶于 lOO L TE中，取 5μί进行 1.0 % 琼脂糖凝股电泳， 以 -EcoT14 I digest DNA Marker为标准， 检测所 得 DNA 的完整性和大小。 用核酸蛋白质分析仪测定 DNA 溶液的 OD26。、 OD28。值以及 OD26。/OD28。比值， 并计算 DNA的浓度 ( g'ml-1) 纯度和 DNA产率 (μ§·^)。
所提取的 DNA作为进行芋螺毒素基因克隆的模板， 用于下面的 PCR扩增。
2. PCR反应及其产物的克隆、 测序、 和序列分析
根据 α-CTx前体基因内含子序列及其 3，端非翻译区 （3，-UTR ) 序列， 设计 α-CTx特异引物。
上游内含子引物序列：
5'-GTGGTTCTGGGTCCAGCA-3' ( SEQ ID NO: 9 ) ； 下游 3，-UTR引物序列：
5'-GTCGTGGTTCAGAGGGTC-3' ( SEQ ID NO: 10 ) 。
每条引物均为 18个^ ^的寡核苷酸片段。
将所提取的基因組 DNA原液稀幹后作为 PCR扩增的模板。 采用 如下的 PCR扩增体系和反应条件：
( 1 ) PCR反应体系：
PCR反应体系 反应体积（ 25μ1 )
模板 DNA 4μ1
P1正向引物 (5μπιο1/μ1) Ιμΐ
PI 反向引物 (5μπιο1/μ1) Ιμΐ
2xTaq PCR MasterMix 12.5μ1
无菌 ddH20 6.5μ1
( 2 ) PCR反应 ：
预变性 94 °C5min 94 °C 7min 变性 94。C 30s
退火 50。C lmin 35 个循环 延伸 72 °C 2min
Figure imgf000035_0001
再延伸 72。C lOmin 回收 PCR特异扩增产物，与 T-easy载体 (Promega)连接后转化大 肠杆菌 XL1菌林（也可以使用其它商业化的感受态大肠杆菌）， 利用 蓝白菌落和氨苄青霉素抗性挑选重組子， 抽提纯化重組子质粒用于测 序分析。 得到测序结果， 如下：
GTGGTTCTGGGTCCA GC4TTTCGTGGCAGGGACGCCGCAGC CAAAGCGTCTGGCCTGGTTGGTCTGACTGACAGGAGAGGATG CTGTTCTCATCCTGCCTGTAACGTAGATCATCCAGAAATTTGT GGCTGA ( SEQ ID NO: 1 ) 。
上面的序列中， 斜体字母是内含子， 对应引物。
所获 PCR特异扩增产物序列经 DNAStar软件分析， 获知其编码 蛋白序列、 3，-非翻译区 （UTR )序列。 经序列分析比较， 获得了本发 明的 1种新型（¾4〃-CTx LvIA/LvD21的前体基因， 即 SEQ ID NO: 1 中带下划线的部分， 其为编码 LvIA/LvD21 芋螺毒素前肽的核苷酸序 列， 如下 （ 114aa ) ：
TTTCGTGGCAGGGACGCCGCAGCCAAAGCGTCTGGCCTG GTTGGTCTGACTGACAGGAGAGGATGCTGTTCTCATCCTGCCT GTAACGTAGATCATCCAGAAATTTGTGGCTGA ( SEQ ID NO: 2 ) 。
根据前体基因及芋螺毒素特点， 推测出 LvIA/LvD21芋螺毒素前 肽， 它具有 SEQ ID NO: 2所示的氛基酸序列 （37aa ) ， 下文中也称 为 α-conotoxin LvIA/LvD21 precursor或 - LvIA/LvD21 precursor 或 LvIA/LvD21 precursor或 LvIA或 LvD21 precursor, 如下：
FRGRDAAAKASGLVGLTDRRGCCSHFAClSyOllFEICGi SEQ ID NO: 3 ) 。
芋螺毒素前体蛋白的信号肽、 前肽以及成熟肽的预测， 采用在线 ProP 1.0 Server进行分析 (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection： PEDS 2004， 77 (1), 107-12.) 。 推断的方法和原理请参考 Luo
S, Zhangsun D, Zhang B， Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006,12(ll):693-704e 推导过程和 结果亦参见图 1。
根据前肽序列再推测出成熟肽 LvIA/LvD21 ,它具有 SEQ ID NO: 4所示的氨基酸序列（16aa;下文中也称为 α-conotoxin LvIA/LvD21 或 - LvIA/LvD21或 LvIA/LvD21或 LvIA或 LvD21 ) ：
GCCSHPACNVDHPEIC# ( SEQ ID NO: 4; #表示 C-末端酰 胺化） 。
LvIA/LvD21含有 α-CTx所特有的 CC-C-C半胱氨酸模式， 二硫 键连接方式 I-III, II-IV (图 2A-B ) , 即在第一和第三个半胱氛酸之间， 以及第二和第四个半胱氨酸之间分别形成两对二硫键。 LvIA/LvD21 为 4/7型 α-CTx (图 1和图 2A-B ) 。
实际上，本发明的成熟肽 LvIA/LvD21也可由前肽 ( SEQ ID NO: 3或 6 )在体内或体外经过相应的加工（例如图 1所示）得到，可选地， 在体内或体外通过酰胺化酶对其 C末端进行酰胺化。
编码 LvIA/LvD21成熟肽的核苷酸序列如下 （ 48 bp ) ：
GGATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCA GAAATTTGT ( SEQ ID NO: 5 ) 。
本发明还涉及成熟肽在未经第二个加工位点（ processing 2 )的序 列 （ 17aa ) ：
GCCSHPACNVDHPEIC G ( SEQ ID NO: 6 ) ；
其对应的核苷酸序列如下 （54bp ) ： GGATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCA GAAATTTGTGGCTGA ( SEQ ID NO: 7 ) ； 或 51bp的
GGATGCTGTTCTCATCCTGCCTGTAACGTAGATCATCCA GAAATTTGTGGC ( SEQ ID NO: 8 ) 。 实施例 -芋螺毒素 TxIB/Txd4基因的克隆和序列分析
1. 织锦芋螺基因組 DNA的提取
分别以从海南岛、 西沙群岛等沿海采集的织锦芋螺（C. textile Linnaeus)活体为材料， 储存在 -80 *€备用。 先将芋螺毒腺解剖出来， 并称重。 然后用海洋动物基因組 DNA提取试剂盒（购自中国北京天 根生化科技有限公司）， 提取毒腺的基因組 DNA, 具体操作参见试剂 盒说明书。 将提取的芋螺基因組总 DNA溶于 ΙΟΟμΙ^ TE中， 取 5μ 进行 1.0%琼脂糖凝股电泳，以 -EcoT14 I digest DNA Marker为标准， 检测所得 DNA的完整性和大小。用核酸蛋白质分析仪测定 DNA溶液 的 OD260、 OD280值以及 OD260/OD280比值， 并计算 DNA的浓度 ( g.mr1) 纯度和 DNA产率 ( g*g-1)。 所提取的完整 DNA用于下一步 PCR扩增进行芋螺毒素基因克隆的模板。
2. PCR反应及其产物的克隆、 测序、 和序列分析
PCR反应的方法、 体系、 条件、 所用引物等参考实施例 1-(1)， 除 了模板为本实施例 1.中所提取的基因組 DNA原液稀释。
回收 PCR特异扩增产物，与 T-easy载体 (Promega)连接后转化大 肠杆菌 XL1菌林（也可以使用其它的商业化的感受态大肠杆菌细胞）， 利用蓝白菌落和氨苄青霉素抗性挑选重組子， 抽提纯化重組子质粒用 于测序分析， 得到 PCR特异扩增产物的序列。
所获 PCR特异扩增产物序列经 DNAStar软件分析， 获知其编码 蛋白序列、 3， -非翻译区（UTR )序列。 经序列分析， 获得了本发明的 1种新型 -CTx TxIB/Txd4的前体基因 (SEQ ID NO: 21) (图 10 ) 。
芋螺毒素前体蛋白的信号肽、 前肽以及成熟肽的预测， 采用在线 ProP 1.0 Server进行分析 (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection： PEDS 2004， 77 (1), 107-12·)。
根据前体基因及芋螺毒素特点，推导出 TxIB/Txd4芋螺毒素前肽， 它具有 SEQ ID NO: 15所示的含有 41 ^酸的蛋白序列。
根据前肽序列再推导出成熟肽 TxIB/Txd4或 TxIB/Txd4(G)，它们 分别具有 SEQ ID NO: 11或 SEQ ID NO: 12所示的氛基酸序列， 推 导的方法和原理请参考 Luo S, Zhangsun D, Zhang B, Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006,12(ll):693-704, 以及在线软件 ProP 1.0 Server。
推导结果详见图 10。
成熟肽都具有 CC-C-C的半胱氨酸模式。 TxIB(G)的 C-末端只比 TxIB 多一个甘氛酸 （ G ) ， 是 TxIB 的类似物。 TxIB/Txd4 或 TxIB/Txd4(G)含有 a -CTx所特有的 CC-C-C半胱氨酸模式，二硫键连 接方式可以为 I-III，II-IV (图 11， A - B ) , 即在第一和第三个半胱氛 酸之间， 以及第二和第四个半胱氨酸之间分别形成两对二硫键。 TxIB/Txd4和 TxIB/Txd4(G)为 4/7型 oc -CTx (图 10和图 11 ) 。
( 1 ) SEQ ID NO: 11 (本文中也称为 oc -conotoxin TxIB/Txd4 或 a - TxIB/Txd4或 TxIB/Txd4或 TxIB )所示的氨基酸序列 （成熟肽）： GCCSDPPCRNKHPDLC ( SEQ ID NO: 11 ) ( 16aa ) 。
优选地， C -末端的半胱氨酸 （ C ) 是酰胺化的， 即表示为 GCCSDPPCRNKHPDLC #， 其中 #表示 C-末端酰胺化。
( 2 ) SEQ ID NO: 12 (本文中也称为 -conotoxin TxIB/Txd4(G) 或 oc - TxIB/Txd4(G)或 TxIB/Txd4(G)或 TxIB(G) )所示的處基酸序列 (成熟肽） ：
GCCSDPPCRNKHPDLC G ( SEQ ID NO: 12 ) ( 17aa ) 。 优选地， C -末端的甘氨酸 （ G ) 是酰胺化的， 即表示为 GCCSDPPCRNKHPDLC G #， 其中 #表示 C-末端酰胺化。 不拘于理论的限制，没有酰胺化的 SEQ ID NO: 12的 C -末端的 甘氛酸（第 17位）可以是酰胺化酶（在细胞内或细胞外）的识别位点， 从而导致与该甘氨酸紧邻的半胱氨酸（C， 第 16位）的酰胺化， 这种 情况下将得到酰胺化的 SEQ ID NO: 11( GCCSDPPCRNKHPDLC # )。
( 3 ) SEQ ID NO: 13所示的 ^酸序列：
GCCSDPPCRNKHPDLC GG ( SEQ ID NO: 13 ) ( 18aa ) 。 不拘于理论的限制， SEQ ID NO: 13的第 18位的甘氨酸可以是 酰胺化酶（在细胞内或细胞外） 的识别位点， 从而导致与该甘氨酸紧 邻的 17位的甘氛酸（ G )的酰胺化， 这种情况下将得到酰胺化的 SEQ ID NO: 12 ( GCCSDPPCRNKHPDLC G # ) 。
或者，
SEQ ID NO: 13的第 17位的甘氨酸可以是酰胺化酶（在细胞内 或细胞外）的识别位点，从而导致与该甘氨酸紧邻的 16位的半胱氛酸 ( C ) 的酰胺化， 这种情况下将得到酰胺化的 SEQ ID NO : 11 ( GCCSDPPCRNKHPDLC # ) 。
( 4 ) SEQ ID NO: 14所示的 ^酸序列：
GCCSDPPCRNKHPDLC GGRR ( SEQ ID NO: 14 ) ( 20aa )。 不拘于理论的限制， SEQ ID NO: 14的第 18位的甘氨酸可以是 酰胺化酶（在细胞内或细胞外） 的识别位点， 从而导致与该甘氨酸紧 邻的 17位的甘氛酸（ G )的酰胺化， 这种情况下将得到酰胺化的 SEQ ID NO: 12 ( GCCSDPPCRNKHPDLC G # ) 。
或者，
SEQ ID NO: 14的第 17位的甘氨酸可以是酰胺化酶（在细胞内 或细胞外）的识别位点，从而导致与该甘氨酸紧邻的 16位的半胱氛酸 ( C ) 的酰胺化， 这种情况下将得到酰胺化的 SEQ ID NO : 11 ( GCCSDPPCRNKHPDLC # ) 。
( 5 ) SEQ ID NO: 15 (本文中也称为 oc -conotoxin TxIB/Txd4 precursor或 oc - TxIB/Txd4 precursor或 TxIB/Txd4 precursor或 TxIB precursor )所示的氛基酸序列 （前体肽） ：
FDGRNTSANNKA TDLMALPVR GCCSDPPCRNKHPDLC GGRR
( SEQ ID NO: 15 ) (41 aa)。
( 6 ) SEQ ID NO: 16所示的核苷酸序列 （编码 TxIB/Txd4成熟 肽） ：
GGATGCTGTTCCGATCCTCCCTGTAGAAACAAGCACCCA GATCTTTGT ( SEQ ID NO: 16 ) ( 48 bp ) 。
( 7 ) SEQ ID NO: 17所示的核苷酸序列 （编码 TxIB/Txd4成熟 肽或编码 TxIB ( G )成熟肽） ：
GGATGCTGTTCCGATCCTCCCTGTAGAAACAAGCACCCA GATCTTTGTGGC ( SEQ ID NO: 17 ) ( 51 bp ) 。
( 8 ) SEQ ID NO: 18所示的核苷酸序列 （编码 TxIB/Txd4成熟 肽或编码 TxIB ( G )成熟肽） ：
GGATGCTGTTCCGATCCTCCCTGTAGAAACAAGCACCCA GATCTTTGTGGCGGA ( SEQ ID NO: 18 ) ( 54 bp ) 。
( 9 ) SEQ ID NO: 19所示的核苷酸序列 （编码 TxIB/Txd4成熟 肽前体或编码 TxIB ( G )成熟肽前体 ) ：
GGATGCTGTTCCGATCCTCCCTGTAGAAACAAGCACCCA GATCTTTGTGGCGGAAGACGCTGA ( SEQ ID NO: 19 ) ( 63 bp )。
( 10 ) SEQ ID NO: 20所示的核苷酸序列 （编码 TxIB/Txd4 或 TxIB ( G )前体蛋白序列 ) ：
TTTGATGGCAGGAATACCTCAGCCAACAACAAAGCGACTG ACCTGATGGCTCTGCCTGTCAGGGGATGCTGTTCCGATCCTCC CTGTAGAAACAAGCACCCAGATCTTTGTGGCGGAAGACGCTG
A ( SEQ ID NO: 20 ) ( 123 bp ) 。
( 11 ) SEQ ID NO: 21所示的核苷酸序列：
GTGGTTCTGGGTCCAGCATTTGATGGCAGGAATACCTCAGC CAACAACAAAGCGACTGACCTGATGGCTCTGCCTGTCAGGGG ATGCTGTTCCGATCCTCCCTGTAGAAACAAGCACCCA GATCTTTGTGGCGGAAGACGCTGATGCTCCAGGACCCTCTGA ACCACGAC (斜体字母是内含子，对应引物）（ SEQ ID NO: 21 ) ( 170 bp ) 。 实施例 1-(3)： α-芋螺毒素 TxIC/Txdl的基因克隆和序列分析
1. 织锦芋螺毒腺基因組 DNA的提取
分别以从海南岛、 西沙群岛等沿海采集的织锦芋螺（C. textile Linnaeus)活体为材料， 储存在 -80。C备用。 先将芋螺毒腺解剖出来， 并称重。 然后用海洋动物基因組 DNA提取试剂盒（购自中国北京天 根生化科技有限公司）， 提取毒腺的基因組 DNA, 具体操作按照试剂 盒说明书进行。 得到毒腺的基因組 DNA。
将提取的毒腺基因組 DNA溶于 lOO L TE中， 取 5μί进行 1.0% 琼脂糖凝股电泳， 以 -EcoT14 I digest DNA Marker为标准， 检测所 得 DNA 的完整性和大小。 用核酸蛋白质分析仪测定 DNA 溶液的 OD26。、 OD28。值以及 OD26。/OD28。比值， 并计算 DNA的浓度 ( g'ml-1) 纯度和 DNA产率 (μ§·^)。
所提取的 DNA作为进行芋螺毒素基因克隆的模板， 用于下面的 PCR扩增。
2. PCR反应及其产物的克隆、 测序、 和序列分析
PCR反应的方法、 体系、 条件、 所用引物等参考实施例 1-(1)， 除 了模板为本实施例 1.中所提取的基因組 DNA原液稀释至终浓度为 3 取 8μ1扩增产物用 1.5%琼脂糖凝胶电泳检测， 电压 90V, 电泳 20min, 以 DL2000 DNA Marker为标准， 检测扩增产物的大小。
回收 PCR扩增产物，与 T-easy载体 (Promega)连接后转化大肠杆 菌 XL1菌林（也可以使用其它商业化的感受态大肠杆菌）， 利用蓝白 菌落和氨苄青霉素抗性挑选重組子， 抽提纯化重組子质粒用于测序分 析。 得到两个测序结果， 即 SEQ ID NO: 22和 SEQ ID NO: 23 (图 14， 168 bp ) ， 分别如下：
Figure imgf000042_0001
GGATGCTGTTCCCATCCTGTCTGTAGCGCGATGAGTCCAATCT GTGGCTGAAGACGCTGATGCTCCAGGACCCTCTGAACCACGA CA ( SEQ ID NO: 22 )或，
GTGGTTCTGGGTCCAGCATTTGATGGCAGGAATGCTGCA GGCAACGACAAAATGTCCGCCCTGATGGCTCTGACCASCAGG GGATGCTGTTCCCATCCTGTCTGTAGCGCGATGAGTCCAATCT GTGGCTGAAGACGCTGATGCTCCAGGACCCTCTGAACCACGA CA ( SEQ ID NO: 23 ) 。
上面的两个序列只有第 77位的 不同， 用加框标出。
所获的 PCR扩增产物的测序结果经 DNAStar软件分析，获知其编 码蛋白序列、 3，-非翻译区 （UTR )序列。 经序列分析比较， 获得了本 发明的新型（x4/6-CTx TxIC/Txdl的前体基因。 即 SEQ ID NO: 22和 SEQ ID NO: 23中带下划线的部分， 其为编码 TxIC/Txdl芋螺毒素前 肽的核苷酸序列， 如下 （114aa ) ：
TTTGATGGCAGGAATGCTGCAGGCAACGACAAAATGTCC
GCCCTGATGGCTCTGACCAgCAGGGGATGCTGTTCCCATCCT GTCTGTAGCGCGATGAGTCCAATCTGTGGCTGA ( SEQ ID NO:
24 )；
TTTGATGGCAGGAATGCTGCAGGCAACGACAAAATGTCC GCCCTGATGGCTCTGACCA@CAGGGGATGCTGTTCCCATCCT GTCTGTAGCGCGATGAGTCCAATCTGTGGCTGA ( SEQ ID NO:
25 ) 。
根据前体基因及芋螺毒素特点，推测出 TxIC/Txdl芋螺毒素前肽， 它具有 SEQ ID NO: 26或 SEQ ID NO: 27所示的氛基酸序列（ 37 aa )， 下文中也称为 α-conotoxin TxIC/Txdl precursor 或 - TxIC/Txdl precursor或 TxIC/Txdl precursor或 TxIC precursor ) ：
FDGRNAAGNDKMSALMALTTRjGCCSHPVCSAMSPIC G ( SEQ ID NO: 26 ) ；
FDGRNAAGNDKMSALMALTIRjGCCSHPVCSAMSPIC G ( SEQ ID NO: 27 ) 。
芋螺毒素前体蛋白的信号肽、 前肽以及成熟肽的预测， 采用在线 ProP 1.0 Server进行分析 (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection： PEDS 2004， 77 (1), 107-12.) 。 推断的方法和原理请参考 Luo
S, Zhangsun D, Zhang B， Quan Y, Wu Y. Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006,12(ll):693-704e 推导过程和 结果亦参见图 14。
根据前肽序列推测出成熟肽 TxIC/Txdl , 它具有 SEQ ID NO: 28 所示的氨基酸序列 （下文中也称为 α-conotoxin TxIC/Txdl 或 α- TxIC/Txdl或 TxIC/Txdl或 TxIC ) ：
GCCSHPVCSAMSPIC # ( SEQ ID NO: 28， #表示 C末端酰胺化， 15aa )
TxIC/Txdl含有 α-CTx所特有的 CC-C-C半胱氨酸模式， 二硫键 连接方式 I-III, II-IV (图 15A ) ， 即在第一和第三个半胱氛酸之间， 以及第二和第四个半胱氨酸之间分别形成两对二硫键。 TxIC/Txdl为 4/6型 α-CTx (图 14和图 15A ) 。 TxIC/Txdl是新的 α-芋螺毒素， 与 其它 α-CTx的序列和活性比较见表 6。
实际上， 本发明的成熟肽 TxIC/Txdl也可由前肽 ( SEQ ID NO: 26或 27或 30 )在体内或体外经过相应的加工（例如图 14所示）得到， 可选地， 在体内或体外通过酰胺化酶对其 C末端进行酰胺化。 编码 TxIC/Txdl成熟肽的核苷酸序列如下 （45 bp ) ：
GGATGCTGTTCCCATCCTGTCTGTAGCGCGATGAGTCCA
ATCTGT ( SEQ ID NO: 29 ) 。
本发明还涉及成熟肽在未经第二个加工位点（ processing 2 )的序 列 （ 16aa ) ：
GCCSHPVCSAMSPIC G ( SEQ ID NO: 30 ) ,
其对应的核苷酸序列如下 （51bp ) ：
GGATGCTGTTCCCATCCTGTCTGTAGCGCGATGAGTCCA ATCTGTGGCTGA ( SEQ ID NO: 31 ) 。 实施例 2-α): α-芋螺毒素 LvIA/LvD21的人工合成
根据 α-芋螺毒素 LvIA/LvD21成熟肽的氛基酸序列（ SEQ ID NO: 4， C末端酰胺化 )，采用 Fmoc方法人工合成 LvIA/LvD21线性肽（图 2B ) 。 具体方法如下：
树脂肽采用 Fmoc化学方法进行人工合成， 可用多肽合成仪或手 工合成法合成树脂肽。 除了半胱氨酸外， 其余氛基酸用标准的侧链保 护基团。 LvIA/LvD21的第 1和第 3个半胱氛酸（ Cys ) 的 -SH用 Trt (S-trityl) 保护， 第 2 和第 4 个半胱氛酸的 -SH 用 Acm (S-acetamidomethyl) 成对保护。其合成步楝为： 采用固相合成法中的 Fmoc 与 FastMoc方法， 在 ABI Prism 433a 多肽合成仪上合成了 3 个异构体线性肽。 Fmoc 氛基酸的侧链保护基为： Pmc (Arg)、 Trt(Cys), But (Thr, Ser、 Tyr)、 OBut (Asp), Boc (Lys)。采用 Fmoc HOBT DCC 方法， Rink酰胺化树脂及 Fmoc 氛基酸，合成步骤参考仪器合成手册 进行。 为反应完全， 在哌啶脱保护及偶合时间上分别适当延长， 对难 接氨基酸采用双偶合， 获得树脂肽。 用 reagent K (trifluoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5： 7.5 : 5 , W W
V / V / v) 将线性肽从树脂上切割下来， 并用冰乙醚沉淀和洗涤回收线 性肽粗品， 用制备型反向 HPLC C18 柱（Vydac) 纯化， 洗脱线性梯 度为在 0― 40min内 0— 40 % B90,第 40— 45 min 40― 100 % B90. 溶 剂 B90 是 90 % ACN ( acetonitrile ) ， 10 % H20， 0.05 % TFA ( trifluoroacetic acid )； 溶剂 A是 0.075 % TFA 的水溶液。 紫外吸收 值分析在 214nm 波长下进行。 纯化后的线性肽用分析型的 HPLC C18 柱（ Vydac )进行纯度检测（图 2C )， HPLC色谱分析^ ^与制备纯化 所用条件相同， 流速为 0.75ml/min， α-芋螺毒素 LvIA/LvD21线性肽的 出峰时间为 27.713min。
参照文献（ Dowell, C; Olivera, Β. Μ·; Garrett, J. Ε·; Staheli, S. Τ·; Watkins, Μ·; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. Μ·; Mcintosh, J. M.， Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience 2003, 23 (24), 8445-52. )对 LvIA/LvD21的线性肽进行两步氧化折叠反应， 过程 简述如下：
首先通过铁氛化钾氧化法（ 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min )在 Trt保护基团的两个半胱氛酸之间形成第一 对二硫键。 单环肽经反相 HPLC C18 柱（Vydac) 纯化后， 进行碘氧 化 ( 10 mM iodine in H20:trifluoroacetic acid: acetonitrile (78:2:20 by volume, 10 min ) , 移去另外 2个半胱氨酸上的 Acm， 同时在这 2个 半胱氨酸之间形成第二对二硫键（图 2B )。二环肽再经反相 HPLC C18 柱（ Vydac) 纯化， 即获得按照从 N端至 C端的顺序在相应的半胱氨 酸之间定向形成二硫键的 α-芋螺毒素， LvIA/LvD21 的出峰时间为 27.947 min (图 2D ) ， 并通过质谱 (MS)鉴定为正确。 HPLC色谱分析 条件为： 用 Vydac C 18 HPLC反相分析柱， 在 40分钟内进行线性梯 度洗脱， B液从 0-40 %， A液从 100 %到 60 %， A液是 0.075 % 的三 氟乙酸（trifluoroacetic acid, TFA) ， B 0.05 % TFA与 90 % 乙 腈 （acetonitrile ) 的水溶液， 流速为 0.75ml/min。 紫外分析波长为 214請。
氧化折叠后的 LvIA/LvD21的理论分子量 (monoisotopic mass)与测 定分子量一致： LvIA/LvD21的理论分子量为 1678.91 Da, LvIA/LvD21 的测定分子量为 1678.7977 Da, 比它的线性肽分子量 1682.91 Da减少 4 Da。 多肽浓度用 280nm波长下比色测定， 根据 Beer-Lambert 方程 ( equation )计算多肽浓度和质量。 这些定量过的折叠好的毒素肽用 于下面实施例中的活性实验。 实施例 2-(2)： -芋螺毒素 ΤχΙΒ与 TxIB ( G ) 的人工合成
根据 -芋螺毒素 TxIB与 TxIB ( G )成熟肽的 ^酸序列 （ SEQ ID NO: 11和 12， C -末端均酰胺化 )，采用 Fmoc方法人工合成 TxIB 与 TxIB ( G ) 线性肽（图 11 ) 。 具体方法如下：
树脂肽采用 Fmoc化学方法进行人工合成， 可用多肽合成仪或手 工合成法合成树脂肽。 除了半胱氨酸外， 其余氛基酸用标准的侧链保 护基团。 TxIB与 TxIB ( G ) 的第 1和第 3个半胱氛酸（ Cys ) 的 -SH 用 Trt (S-trityl) 保护， 第 2 和第 4 个半胱氛酸的 -SH 用 Acm (S-acetamidomethyl) 成对保护。其合成步楝为： 采用固相合成法中的 Fmoc 与 FastMoc方法， 在 ABI Prism 433a 多肽合成仪上合成了 3 个异构体线性肽。 Fmoc 氛基酸的侧链保护基为： Pmc (Arg)、 Trt(Cys), But (Thr、 Ser、 Tyr),OBut (Asp),Boc (Lys).采用 Fmoc HOBT DCC方 法， Rink酰胺化树脂及 Fmoc 氛基酸， 合成步骤参考仪器合成手册进 行。 为反应完全，在哌啶脱保护及偶合时间上分别适当延长，对难接氨 基酸采用默偶合， 获得树脂肽。 用 reagent K (trittuoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5： 7.5： 5,v / v / v / v / v) 将线性肽从树脂上切割下来， 并用冰乙醚沉淀和洗涤回收线性 肽粗品， 用制备型反向 HPLC C18 柱（Vydac) 纯化， 洗脱线性梯度 为在 0-40min内 2-42% B60, 42-47 min 42-100% B60。 溶剂 B60是 60% ACN( acetonitrile ) , 40 % H20, 0.05% TFA( triHuoroacetic acid )； 溶剂 A 0.05% TFA 的水溶液。
纯化后的线性肽用分析型的 HPLC C18 柱（Vydac )进行纯度检 测， 洗脱梯度为 0 - 40 min 2 - 42% B60, 42-47 min 42 - 100% B60,流 速为 l mL/min。 其纯度达 95 %以上， 用于氧化折叠。
参照文献（ Dowell, C; Olivera, Β. Μ·; Garrett, J. Ε·; Staheli, S. Τ·; Watkins, Μ·; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. Μ·; Mcintosh, J. M.， Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience 2003, 23 (24)， 8445-52. )对 TxIB与 TxIB ( G )的线性肽进行两步氧化折叠反应， 过程简述如下：
首先通过铁氛化钾氧化法（ 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min )在 Trt保护基团的两个半胱氛酸之间形成第一 对二硫键。 单环肽经反相 HPLC C18 柱（Vydac) 纯化后， 进行碘氧 化 ( 10 mM iodine in H20:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min ) ， 移去另外 2个半胱氛酸上的 Acm， 同时在这 2个 半胱氨酸之间形成第二对二硫键。 二环肽再经反相 HPLC C18 柱 ( Vydac) 纯化， 即获得按照从 N端至 C端的顺序在相应的半胱氛酸 之间定向形成二硫键的（X -芋螺毒素， 并通过质谱 (MS)鉴定为正确。
氧化折叠后的 TxIB与 TxIB( G )的理论分子量 (monoisotopic mass) 与测定分子量一致： TxIB的理论分子量为 1738.7 Da, TxIB的测定分子 量为 1738.6 Da; TxIB ( G )的理论分子量为 1795.7 Da; TxIB ( G )的 测定分子量为 1795.6 Da。 多肽浓度用 280nm 波长下比色测定， 根据 Beer-Lambert方程（ equation )计算多肽浓度和质量。这些定量过的折 叠好的毒素肽用于后续的活性实验。 实施例 2-ί3): α-芋螺毒素 TxIC的人工合成
根据 α-芋螺毒素 TxIC成熟肽的氛基酸序列 （SEQ ID NO: 28， C末端酰胺化 ) ， 采用 Fmoc方法人工合成 TxIC线性肽（图 15A ) 。 具体方法如下：
树脂肽采用 Fmoc化学方法进行人工合成， 可用多肽合成仪或手 工合成法合成树脂肽。 除了半胱氨酸外， 其余氛基酸用标准的侧链保 护基团。 TxIC的第 1和第 3个半胱氛酸（ Cys )的-SH用 Trt (S-trityl) 保护， 第 2和第 4个半胱氛酸的 -SH用 Acm (S-acetamidomethyl) 成 对保护。其合成步骤为：采用固相合成法中的 Fmoc 与 FastMoc方法， 在 ABI Prism 433a 多肽合成仪上合成了 3个异构体线性肽。 Fmoc 氨 基酸的侧链保护基为： Pmc (Arg)、 Trt(Cys), But (Thr、 Ser、 Tyr)、 OBut (Asp), Boc (Lys).采用 Fmoc HOBT DCC方法， Rink酰胺化树 脂及 Fmoc氨基酸， 合成步骤参考仪器合成手册进行。 为反应完全， 在哌啶脱保护及偶合时间上分别适当延长，对难接氨基酸采用默偶合， 获得树脂狀。 用 reagent K (trifluoroacetic acid /water / ethanedithiol / phenol / thioanisole; 90 : 5 : 2.5 : 7.5： 5 , v / v / v / v / v) 将线性肽从树 脂上切割下来， 并用冰乙醚沉淀和洗涤回收线性肽粗品， 用制备型反 向 HPLC C18 柱（ Vydac) 纯化， 洗脱线性梯度为在 0 - 40min内 15 - 50% B90, 第 40 - 45 min 50 - 100% B90. 溶剂 B90是 90% ACN ( acetonitrile ) ， 10 % H20, 0.5% TFA ( trifluoroacetic acid ) ； 溶剂 A是 0.65% TFA 的水溶液。
紫外吸收值分析在 214nm 波长下进行。 纯化后的线性肽用分析型 的 HPLC C18柱（ Vydac )进行纯度检测，洗脱梯度为 0—40 min 2-42% B60, 42 - 47 min 42-100% B60， 流速为 1 mL/min。 其纯度达 95 % 以上， 用于氧化折叠。
参照文献（ Dowell, C; Olivera, Β. Μ·; Garrett, J. Ε·; Staheli, S. Τ·; Watkins, Μ·; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. Μ·; Mcintosh, J. M.， Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience 2003, 23 (24)， 8445-52. )对 TxIC的线性肽进行两步氧化折叠反应， 过程简述如 下：
首先通过铁氛化钾氧化法（ 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min )在 Trt保护基团的两个半胱氨酸之间形成第 一对二硫键。 单环肽经反相 HPLC C18 柱（Vydac) 纯化后， 进行碘 氧化 ( 10 mM iodine in H20:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min ) ， 移去另外 2个半胱氛酸上的 Acm， 同时在这 2 个半胱氨酸之间形成第二对二硫键。 二环肽再经反相 HPLC C18 柱 ( Vydac) 纯化， 洗脱线性梯度仍为在 0 - 40min内 15-50% B90, 第 40 - 45 min 50 - 100% B90. 溶剂 B90是 90% ACN ( acetonitrile ) , 10 % H20, 0.5% TFA( trifluoroacetic acid ); 溶剂 A是 0.65% TFA 的 水溶液。 紫外吸收值分析在 214nm 波长下进行。 即获得按照从 N端至 C 端的顺序在相应的半胱氨酸之间定向形成二硫键的 α-芋螺毒素， TxIC的出峰时间为 23.366min (图 15B ) ， 并通过质谱 (MS)鉴定为正 确。
氧化折叠后的 TxIC的理论分子量 (monoisotopic mass)与测定分子 量一致： TxIC 的理论分子量为 1488.81 Da, TxIC 的测定分子量为 1488.4266 Da, 比它的线性肽分子量 1492.815 Da减少 4 Da。 多肽浓 度用 280nm波长下比色测定， 根据 Beer-Lambert 方程 ( equation ) 计算多肽浓度和质量。 这些定量过的折叠好的毒素肽用于下面实施例 中的活性实验。 实施例 3-α): α-芋螺毒素 LvIA/LvD21阻断各种 nAChRs的实验 参照文献 ( 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. ) 中的方 法， 以及体夕卜转录试剂盒 ( mMessage mMachine in vitro transcription kit (Ambion，Austin，TX) )说明书， 制备各种大鼠神经型 nAChRs 亚 型 （α3β2， «6/α3β2β3, «6/α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7 )、人类 α3β2, «6/α3β2β3 , α3β4、 以及小鼠肌肉型 nAChRs ( αΐβΐδε ) 的 cRNA, 其浓度用 UV 260 nm下的 OD值进行测算。 解 剖收集非洲爪蟾 ( Xenopus laveis )卵母细胞（蛙卵）， 将 cRNA注射 入蛙卵中，每个亚基的注射量为 5ng cRNA。肌肉 nAChR每个亚基注 射 0.5 - 2.5 ng DNA。 蛙卵在 ND-96中培养。 蛙卵收集后的 1 - 2天内 注射 cRNA, 注射后 1 - 4天内用于 nAChRs的电压钳记录。
将 1个注射过 cRNA的蛙卵置于 30uL的 Sylgard记录槽中 （直 径 4mmx深度 2mm ) ， 重力灌注含有 0.1 mg/ml BSA (bovine serum albumin)的 ND96灌流液（ 96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2， 1.0 mM MgCl2， 5 mM HEPES, pH 7.1― 7.5) 或含有 1 mM atropine 的 ND96 (ND96A) , 流速为 lml/min。 所有的芋螺毒素溶液也含有 0.1 mg/ml BSA 以减少毒素的非特异性吸附， 用转换阀（SmartValve, Cavro Scientific Instruments, Sunnyvale, CA)可以在灌注毒素或乙醜 胆碱 (ACh)之间进行自由切换，以及一系列三通螺线阀（ solenoid valves model 161T031, Neptune Research, Northboro, MA) 使灌注 D96与 ACh 等之间进行自由切换。 Ach 门控的电流由双电极电压箝放大器 ( model OC-725B, Warner Instrument Corp., Hamden, CT )设置在 "慢，，箝， 以及 clamp gain在最大值 ( x2000 )位置时进行在线记录。 用 1mm夕卜径 xO.75 内径 mm的玻璃毛细管 ( fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL )拉制玻璃电极， 并充满 3 M KCl 作为电压和电流电极。 膜电压箝制在 -70mV.整个系统均由电脑控制和 记录数据。 ACh脉冲为每隔 5min 自动灌注 Is的 ACh。 ACh的浓度 分别为，表达肌肉型的 nAChRs 和神经型 α9α10 nAChRs卵为 10μΜ; 表达神经型的 nAChRs之 α7为 200μΜ， 其它的亚型都为 100 μΜ。 至 少记录 4个卵表达某个亚型对不同毒素浓度的电流反应情况， 以及电 流轨迹。
测试的电流数据用 GraphPad Prism软件 (San Diego, CA)进行统 计分析， 绘制剂量反应曲线， 计算芋螺毒素的半阻滞浓度 IC5。等多种 有关毒素阻断 nAChRs的各种参数。
结果表明， LvIA/LvD21 (实施例 2-(1)制备）对大鼠 α3β2 nAChRs 等有阻断作用， 且洗脱较快（图 3 ) 。 LvIA/LvD21对 α3β2 nAChRs 的阻断活性最强， 其半阻断剂量 IC5。仅为 8.69 nM, 误差范围为 6.9 - 11.0 n (表 1 ) 。 100 nM a-LvIA/LvD21完全阻断了由 Ach门控的 大鼠 α3β2 nAChRs 开放产生的电流， 2 min内可洗脱完全， 其阻断是 可逆的（图 3A ) 。 LvIA/LvD21对 α6/α3β4 nAChRs的阻断活性次之， 其半阻断剂量 IC50和误差范围为 120.9 (86.1-169.8) nM ；再次为 α3β4， 其半阻断剂量 IC5。和误差范围为 148.4 (103.2-213.2) nM。 LvIA/LvD21 对（¾6/α3β2β3 nAChRs的阻断活性 f艮微弱， 其半阻断剂量 IC5。和误差 范围为 852(590-1230) nM; 对 α7、 α2β4有极其微弱的阻断活性， 其半 阻断剂量 IC5o和误差范围分别高达为 3000 (1797-4997) nM与 15520 (11600-20770) nM。对其它亚型没有阻断活性，包括 α9α10、 α2β2、 α4β2、 α4β4与 Μαΐβΐδε,其 IC5。 > 10μΜ(表 1 )。 LvIA/LvD21对各种 nAChRs 亚型的剂量反应曲线分别如图 3B、 3C、 3D所示。
相比之下， a-LvIA/LvD21 阻断 α3β2比阻断（¾6/α3β2β3的活性 > 100倍， 在大鼠上高达〜 100倍， 在人类上更是高达〜 305倍（图 3Β, 3D, 表 1 )。 a-LvIA/LvD21是至今发现的第一个对 α3β2 vs. «6/α3β2β3 的选择性和区分度最好的配体。 以前发现的所有阻断 α3β2的芋螺毒素 几乎都同时阻断（¾6/a3p2p3 iiAChRs。 因而， a-LvIA/LvD21是我们发现 的一个真正的 α3β2* vs. α6β2* nAChRs高选择性新阻断剂，对于综合研 究理解该亚型在正常和疾病状态下的功能和意义具有非常重要的价值。 a-LvIA/LvD21选择性阻断对 α3β2 nAChRs的阻断选择性高。 从 100 nM a-LvIA/LvD21 对 α3β2 nAChRs (图 4A )，以及 10 μΜ a-LvIA/LvD21 对其接近的 α2β2 (图 4Β)和 Μα1β1δεα7(图 4C) nAChRs 的电流影响情况可以看出（图 4 )， 100 nM a-LvIA/LvD21完全阻断 α3β2 nAChRs (图 4A ) ， 而比之高 100倍浓度的毒素对 α2β2， 与 Μαΐβΐδε nAChRs亚型没有阻断活性 (图 4B-C)。 因此， a-LvIA/LvD21是本发明人发现的， 对 α3β2 nAChRs活性 很强的新型 α-芋螺毒素， 是第一个对 α3β2 vs. «6/α3β2β3 的选择性和 区分度最好的配体。 表 1: a-LvIA/LvD21对各种 nAChRs亚型的 IC5。和剂量反应曲 线斜率
Figure imgf000052_0001
a Numbers in parentheses are 95% confidence intervals; 表中 a是置信度为 95 %的区间 . b nAChR subtype IC5。 / α3β2 IC5。； b是其它亚型与 a3卩 2 nAChRs半阻断剂量（ IC50 )的比值. c nAChR subtype IC50 I Human α3β2 IC50; c是人类 α6/3卩 2卩 3亚型与人类 α3卩 2 nAChRs半阻断剂量 ( IC50 ) 的比值.
"M" denotes mouse subunits used, "H" denotes human subunits used. "M" <f¾ ^小 [！， "H" -f ^ - 类.
已有研究表明， α3β2、 α6/α3β4与 α3β4 nAChR是治疗神经精神 疾病， 如神经痛、 成瘾、 帕金森病、 痴呆、 精神分裂症、 抑郁、 恐惧等 的药物作用靶点 （参见背景技术中的相关文献） 。 因此， 本发明的新的 α-芋螺毒素 LvIA/LvD21在上述疾病的机理研究、 诊断、 治疗方面具 有极高的应用潜力。 实施例 3-(2)： (X-芋螺毒素 ΤχΙΒ与 TxIB (G)特异阻断 (X 6/ (X 3 β 2 β 3 nAChRs实验
参照文献 ( 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. ) 中的方 法， 以及体夕卜转录试剂盒 ( mMessage mMachine in vitro transcription kit(Ambion，Austin，TX))说明书， 制备各种大鼠神经型 nAChRs 亚 型， 3β2, 6/ 3β2β3 (即（¾6p2*-iiAChRs ) ， 6/ 3β 4, 9 10, 4 β 2, 4 β 4, 3 β 4, 2 β 2, 2 β 4, ΐ) 、 人类 α 6/ 3β2β3, 以及小鼠肌肉型 nAChRs ( oc 1 β 1 δ ε )的 cR A, 其 浓度用 UV 260 nm下的 OD值进行测算。解剖收集非洲爪蟾（ Xenopus laveis )卵母细胞（蛙卵）， 将 cRNA注射入蛙卵中， 每个亚基的注射 量为 5ngcRNA。肌肉 nAChR每个亚基注射 0.5-2.5 ng DNA。蛙卵在 ND-96中培养。 蛙卵收集后的 1-2天内注射 cRNA, 注射后 1 - 4天 内用于 nAChRs的电压钳记录。
将 1个注射过 cRNA的蛙卵置于 30uL的 Sylgard记录槽中 （直 径 4mm X深度 2mm ) ， 重力灌注含有 0.1 mg/ml BSA (bovine serum albumin)的 ND96灌流液（ 96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2， 1.0 mM MgCl2, 5 mM HEPES, pH 7.1-7.5) 或含有 1 mM atropine 的 ND96 (ND96A) , 流速为 lml/min。 所有的芋螺毒素溶液也含有 0.1 mg/ml BSA 以减少毒素的非特异性吸附， 用转换阀（SmartValve, Cavro Scientific Instruments, Sunnyvale, CA)可以在灌注毒素或乙醜 胆碱 (ACh)之间进行自由切换，以及一系列三通螺线阀（ solenoid valves model 161T031, Neptune Research, Northboro, MA) 使灌注 D96与 ACh 等之间进行自由切换。 Ach 门控的电流由双电极电压箝放大器 ( model OC-725B, Warner Instrument Corp., Hamden, CT )设置在 "慢"箝， 以及 clamp gain在最大值 ( x 2000 )位置时进行在线记录。 用 1mm外径 X 0.75内径 mm的玻璃毛细管 （ fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL )拉制玻璃电极， 并充满 3 M KCl 作为电压和电流电极。 膜电压箝制在 -70mV.整个系统均由电脑控制和 记录数据。 ACh脉冲为每隔 5min 自动灌注 Is的 ACh。 ACh的浓度 分别为，表达肌肉型的 nAChRs 和神经型（X 9 (X 10 nAChRs卵为 10 μ Μ; 表达神经型的 nAChRs之 (X 7为 200 μ Μ，其它的亚型都为 100 μ Μ。 至少记录 4个卵表达某个亚型对不同毒素浓度的电流反应情况， 以及电流轨迹。
测试的电流数据用 GraphPad Prism软件 (San Diego, CA)进行统 计分析， 绘制剂量反应曲线， 计算芋螺毒素的半阻滞浓度 IC5。等多种 有关毒素阻断 nAChRs的各种参数。
结果表明， a -TxIB 和 TxIB ( G ) (实施例 2-(2)制备 )对大鼠 (X 6/ oc 3 P 2 P 3 nAChR均有特异阻断作用， 洗脱都 f艮快（图 12 ) . 1 μ Μ a -TxIB/Txd4 几乎完全阻断了由 Ach 门控的大鼠 oc 6/ oc 3 β 2 β 3 nAChR开放产生的电流，洗脱很快， 阻断是可逆的 （图 12A ) 。 相比 之下， oc -TxIB 比 TxIB ( G )的活性要强 8.7倍（图 12B ) ， 它们对 «6/α3β2β3 nAChRs 的半阻断剂量 IC50 和误差范围分别为（X -TxIB, 28.4 (18.6-43.4) ηΜ; - TxIB ( G ) , 247.4 (186.2-328.8) nMe 它们的剂 量反应曲线的斜率 （ Hillslope ) 和误差范围分别为 oc -TxIB, 0.51 (0.41-0.60) 和 oc- TxIB (G) , 0.78 (0.63-0.93). 因此， -TxIB和 TxIB (G)对其它 nAChRs亚型没有阻断活性， 其 IC50 > 10 μ M (图 12C, 表 2) 。
表 2: a -TxIB与 TxIB(G)对各种 nAChRs
亚型的半阻断剂量 IC50和剂量反应曲线的斜率
Figure imgf000055_0001
表 2中 a是置信度为 95%的区间。 b 是 TxIB(G)与 TxIB 半阻断 剂量（IC50) 的比值。 c 是在 10 μΜ下没有阻断活性。
a -TxIB/Txd4 ^ 6/ 3β2β3 nAChR的阻断选择性特高。 从 1 μΜ a -TxIB/Txd4 对（χ 6/(χ 3 β 2 β 3 nAChR , 以及 10 μΜ -TxIB/Txd4 对其非常接近的（Χ3 β 2 ( Β ) ， «6/a3p4(C), 3 β 4(D) nAChRs的电流影响情况可以看出 （图 13) ， 1 μΜ oc -TxIB/Txd4特 ^-ΡΒ.¾Γ 6/ 3β2β3 nAChR (图 13A) ， 而比之高 10倍浓度的毒素 对 oc 3 P 2 (图 13B )， «6/α3β4(® 13C), 与 oc 3 β 4(图 13D) nAChRs亚型 ¾：有任何阻断活性。 对人类 6/ 3β2β3 nAChR, -TxIB和 TxIB (G)具有与大鼠 nAChR相似的阻断活性。 因此， a -TxIB是目前发现的， 对 nAChR选择性最好的 α -芋螺 毒素， 其活性比较见下面的表 3。
已有的研究表明， （X6/0C3P2P3 nAChR是治疗神经精神疾病， 如烟碱、 吗啡与可卡因等的成瘾、 帕金森病、 痴呆、 精神分裂症、 抑郁 等的药物作用靶点 （参见背景技术中的文献） 。 因此， 本发明的新 (X- 芋螺毒素 TxIB/Txd4和 TxIB (G)在上述疾病的机理研究、 诊断、 治 疗方面具有极高的应用价值。
TxIB/Txd4和 TxIB/Txd4(G)与其它 oc-CTx的序列和活性比较见 表 3。
表 3: α-ΤχΙΒ与其它 α-芋螺毒素前体蛋白序列及其活性比较
Figure imgf000056_0001
Figure imgf000057_0001
表中星号 （* )表示 C末端酰胺化。 短线 （- )表示缺口。 实施例 3-ί3) : α-芋螺毒素 TxIC 特异阻断 α3β4 和 α6/α3β4 nAChRs实验
参照文献 ( 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. ) 中的方 法， 以及体夕卜转录试剂盒 ( mMessage mMachine in vitro transcription kit (Ambion，Austin，TX) )说明书， 制备各种大鼠神经型 nAChRs 亚 型 ( α3β4, «6/α3β4, α9α10, α4β2, α4β4, α3β4, α2β2 , α2β4, α7 )、 人类 α3β4、 以及小鼠肌肉型 nAChRs ( αΐβΐδε ) 的 cRNA, 其浓度 用 UV 260 nm下的 OD值进行测算。 解剖收集非洲爪蟾 ( Xenopus laveis )卵母细胞（蛙卵）， 将 cRNA注射入蛙卵中， 每个亚基的注射 量为 5ng cRNA。 肌肉 nAChR每个亚基注射 0.5 - 2.5 ng DNA。 蛙卵 在 ND-96中培养。 蛙卵收集后的 1 - 2天内注射 cRNA, 注射后 1 - 4 天内用于 nAChRs的电压钳记录。
将 1个注射过 cRNA的蛙卵置于 30uL的 Sylgard记录槽中 （直 径 4mmx深度 2mm ) ， 重力灌注含有 0.1 mg/ml BSA (bovine serum albumin)的 ND96灌流液（ 96.0 mM NaCl, 2.0 mM KC1， 1.8 mM CaCl2， 1.0 mM MgCl2， 5 mM HEPES, pH 7.1― 7.5) 或含有 1 mM atropine 的 ND96 (ND96A) , 流速为 lml/min。 所有的芋螺毒素溶液也含有 0.1 mg/ml BSA 以减少毒素的非特异性吸附， 用转换阀（SmartValve, Cavro Scientific Instruments, Sunnyvale, CA)可以在灌注毒素或乙醜 胆碱 (ACh)之间进行自由切换，以及一系列三通螺线阀（ solenoid valves, model 161T031, Neptune Research, Northboro, MA) 使灌注 D96与 ACh 等之间进行自由切换。 Ach 门控的电流由双电极电压箝放大器 ( model OC-725B, Warner Instrument Corp., Hamden, CT )设置在 "慢，，箝， 以及 clamp gain在最大值 ( x2000 )位置时进行在线记录。 用 1mm夕卜径 xO.75 内径 mm的玻璃毛细管 ( fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL )拉制玻璃电极， 并充满 3 M KCl 作为电压和电流电极。 膜电压箝制在 -70mV.整个系统均由电脑控制和 记录数据。 ACh脉冲为每隔 5min 自动灌注 Is的 ACh。 ACh的浓度 分别为，表达肌肉型的 nAChRs 和神经型 a9al0 nAChRs卵为 ΙΟμΜ; 表达神经型的 nAChRs之 α7为 200μΜ， 其它的亚型都为 100 μΜ。 至 少记录 4个卵表达某个亚型对不同毒素浓度的电流反应情况， 以及电 流轨迹。
测试的电流数据用 GraphPad Prism软件 (San Diego, CA)进行统 计分析， 绘制剂量反应曲线， 计算芋螺毒素的半阻滞浓度 IC5。等多种 有关毒素阻断 nAChRs的各种参数。
结果表明， TxIC (实施例 2-(3)制备）对大鼠 α3β4 nAChR均有 特异阻断作用， 洗脱较快（图 16 ) 。 TxIC是迄今为止发现的 α3β4 nAChR的活性最强的阻断剂， 其半阻断剂量 IC5。仅为 12.5 nM， 与其 它已知的芋螺毒素的活性比较见下面的表 4。
1 μΜ α-TxIC/Txdl完全阻断了由 Ach门控的大鼠 α3β4 nAChR 开放产生的电流， 洗脱较快， 阻断是可逆的（图 16A ) 。 TxIC对 α3β4 nAChR的阻断活性最强， 其半阻断剂量 IC5。和误差范围为 12.5 nM (9.4 - 16.5 nM); TxIC对 α6/α3β4 nAChR的阻断活性次之， 其半阻 断剂量 IC50和误差范围为 94.1 nM (73 - 121 nM); TxIC 对 α2β4 nAChR的阻断活性 f艮微弱，其半阻断剂量 IC5()和误差范围为 4550 nM (3950-5230 nM)e TxIC对它们的剂量反应曲线的斜率 （ Hillslope )和 误差范围分别为： α3β4 nAChR, 0.19 (0.66 - 1.44); «6/α3β4 nAChR, 0.26 (0.73― 1.87)； α2β4 nAChR, 0.20 (1.48― 2.42)。 α-TxIC对其它 nAChRs亚型没有阻断活性，包括 α4β4、(χ4β2、(χ6/α3β2β3、α2β2、(χ9α10、 α7、 αΐβΐδε, 其 Ι< 5。> 10μΜ (图 16Β， 表 5 )， 相比之下， α-TxIC 阻 断 α3β4比阻断（¾6/α3β4的活性要强 7.5倍， 比阻断 α2β4的活性要强 524倍（图 16B, 表 5 ) 。
α-TxIC/Txdl 对 α3β4 nAChR 的阻断选择性高。 从 1 μΜ α-TxIC/Txdl 对 α3β4 nAChR, 以及 10 μΜ α-TxIC/Txdl 对其非常接 近的 α4β4 (Β)， （¾7(C) nAChRs的电流影响情况可以看出 （图 17 ) ， 1 μΜ α-TxIC/Txdl特异阻断 α3β4 nAChR (图 17A )， 而比之高 10倍浓 度的毒素对 α4β4(图 17B), 与 α7(图 17C) nAChRs亚型没有阻断活性。 对人类 α3β4 nAChR, α-TxIC具有与大鼠 α3β4 nAChR相似的阻断活 性。
因此， α-TxIC是目前发现的对 α3β4 nAChR活性最强的 α-芋螺毒 素， 同时对（¾6/a3p4 iiAChR也有较强的阻断活性， 其活性比较见下面 的表 4。
表 4: α-TxIC与其它 α-芋螺毒素序列及其活性比较
Figure imgf000059_0001
C.purpurasce RDPCCSNPVCTVHN α6/α3β2β3 (1-1.7 nM) > α6/α3β4(12.6-30.5
PIA
ns PQIC* ηΜ)>α3β2(74.2 nM)> α3β4(518 nM)
GCCSDPRCNYDHPEI α9α10 (19 nM) > α6/α3β2β3 (140 nM) > α6/α3β4
Vcl.l C. victoriae
C* (980 nM) > α3β4 (4200 nM) > α3β2 (7300 nM)
GCCSHPACAGNNQH
GIC C.geographus α3β2 (1.1 nM) > α4β2 (309 nM) > α3β4 (755 nM)
IC*
表中星号 （* )表示 c末端酰胺化。 短线 （- )表示缺口。 表 5: a-TxIC对各种 nAChRs亚型
的半阻断剂量 IC5。和剂量反应曲线的斜率
Figure imgf000060_0001
-中 a是置信度为 95%的区间； b 是其它亚型与 α3β4 nAChR半 量（ ICSQ ) 的比值； c是在 10 μΜ下没有阻断活性。
现有技术的研究表明， α3β4、 （¾6/a3p4 iiAChRs是治疗神经精神疾 病， 如烟碱、 吗啡与可卡因等的成瘾、 神经痛、 帕金森病、 痴呆、 精神 分裂症、抑郁、恐惧等的药物作用靶点 （参见背景技术中的相关文献） 。 因此， 本发明的新的 α-芋螺毒素 TxIC/Txdl在上述疾病的机理研究、 诊断、 治疗方面具有极高的应用价值。 实施例 4: α-芋螺毒素 LvIA/LvD21阻断 α3β2 nAChRs突变型的 实验
α-CTx LvIA/LvD21 对 α3β2 nAChR 的 7 个 β2 突变型： α3β2[Τ59Κ] , α3β2[Τ59ί] , α3β2[Τ59Ι] , α3β2 [Villi] , «3p2[F119Q], α3β2 [Q34A] , α3β2 [Κ79Α】的阻断作用有较大差异 （表 6-7; 图 5-6 )， 这 7种突变型是将 nAChR的 β2亚基中与配体结合部位的关键氛基酸 残基突变为 β4亚基中相应的 ^酸残基 (包括 α-CTx MII在内）。突变 型的具体制备方法参照文献 Shiembob DL, Roberts RL, Luetje CW, Mcintosh JM. Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry. 2006 Sep 19;45(37):11200-7进行。
关于前 5 个 α3β2 nAChRs 突变型的详细情况可参考文献 Shiembob DL, Roberts RL, Luetje CW, Mcintosh JM. Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry. 2006 Sep 19;45(37):11200-7； 和 Dutertre S, Nicke A, Lewis RJ. β2 subunit contribution to 4/7 α-conotoxin binding to the nicotinic acetylcholine receptor. J Biol Chem 2005;280:30460-8。
其中后两个 α3β2 nAChRs 突变型 α3β2 Q34A, α3β2 Κ79Α是 α-CTx LtlA与 α3β2受体结合的关键氛基酸（ Luo， S., Akondi, Κ. Β·， Zhangsun, D., Wu, Υ·， Zhu, Χ·， Hu, Υ·， Christensen, S.， Dowell, C" Daly, N. L., Craik, D. J., Wang, C. I., Lewis, R. J., Alewood, P. F., and Michael Mcintosh, J. (2010) Atypical alpha-conotoxin LtlA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor. J. Biol. Chem.285, 12355-12366 ) 。
具体实验方法可以参考实施例 3-(1)，结果如表 6-7和图 5-6所示。 从表 2和图 5可以看出， α-CTx LvIA/LvD21对突变型 α3β2[νΐ11Ι】 的阻断活性最小，其 IC5。 为 126 nM,其活性比对野生型 α3β2 nAChR ( IC50 为 14.5 nM )的阻断能力下降了 8.7倍。对突变型 a3p2 [F119Q】、 α3β2[Τ59Κ] , α3β2 [T59L]的阻断活性很强， 其 IC5。分别仅为 0.58、 0.96和 2.03 nM, 其活性比对野生型 α3β2 nAChR的阻断能力分别增 强了 25倍、 15倍和 7倍。 α-CTx LvIA/LvD21对突变型 a3p2[Q34A】、 [K79A]与 [T59I]的 IC5。分别为 8.64、 10.8和 15.2 nM, 其阻断活性是 对野生型 α3β2 nAChR的阻断能力 0.6 - 1.05倍，与对野生型的 α3β2 nAChR 的阻断活性差异不大。 α-CTx LvIA/LvD21 对突变型 (¾3P2[F119Q】的阻断活性是对 α3β2[νΐ11Ι】的 217倍。 这意味着 β2亚 基上的第 111位的缬氨酸、第 119位的苯丙氨酸、第 59位的苏氨酸对 于 LvIA与 α3β2的结合起关键作用， 其活性变化趋势有减弱和增强 2 种情况，这与 MII、LtIA和其它以前发现的 α-CTxs结合 α3β2 nAChRs 的部位有所不同。
表 6: a-LvIA/LvD21对 α3β2 nAChRs
野生型及其突变型的 IC5。和剂量反应曲线斜率
Figure imgf000062_0001
上表中，
a是置信度为 95 %的区间； b 是 α3β2 nAChRs突变型与野生型半 阻断剂量（IC5() ) 的比值； c α3β2 nAChRs其它突变型、 野生型与突 变型（¾3P2[F119Q】半阻断剂量（ IC5。） 的比值。
α-CTx LvIA/LvD21不但对有些 α3β2 nAChRs突变型的阻断活性 ( IC50 )有很大的影响， 对它们的洗脱速率的影响也很显著（图 6与表 7 )。研究结果显示， 10 nM a-LvIA/LvD21阻断 α3β2 nAChRs野生型 大约 50 %的电流， 洗脱速度快， 2 min内电流完全恢复（图 6A )； 10 nM a-LvIA/LvD21却阻断了突变型 a3p2[F119Q】的全部电流， 其洗脱 速度慢， 洗脱 12 min后电流才恢复（图 6B ) ；更加不同的是， 10 nM a-LvIA/LvD21阻断了突变型 α3β2[Τ59Κ】的全部电流， 洗脱速度非常 慢， 洗脱 20 min后的电流才恢复至对照电流的 27 % (图 6C )； 然而， 10 nM a-LvIA/LvD21完全不阻断突变型（¾3卩2[ 1111】的电流（图 6D )。
«-LvIA/LvD21 对各种突变型受体的洗脱速率的影响总结于表 7。 α3β2[Κ79Α], α3β2[νΐ11Ι], «3p2[Q34A]， 以及 α3β2[Τ59Ι】 4种突变型对 a-LvIA/LvD21阻断后的洗脱速度影响较小， 在 10 - ΙΟΟΟΟηΜ很宽的 浓度范围类， 它们的洗脱速度都较快， 在 1 - 3 min内， 电流均可恢复 到对照水平， 即 100 %。 对于突变型 （¾3P2[T59L】， 其洗脱速度相对较 慢， 需要 5 - 8 min的洗脱， 其电流方可恢复到对照水平。 对于突变型 (¾3P2[F119Q】来说， 其洗脱速度更慢， 需要 10 - 12 min的洗脱， 其电 流才能恢复到对照水平。 对于突变型 α3β2[Τ59Κ】， 其洗脱速度是最慢 的， 10 nM LvIA/LvD21完全阻断了它的电流， 20 min的洗脱只能恢 复到对照电流的 28±3.5 %， 100 nM LvIA/LvD21阻断后， 20 min的洗 脱仅恢复到对照电流的 13±2 %， 可见突变型 α3β2[Τ59Κ】对 LvIA/LvD21的结合方式是影响最大的。 因此， α-CTx LvIA/LvD21的 结构与功能为研究洞察 α-CTxs与 nAChRs之间相互作用的机制奠定 了重要的基础， 提供了很好的工具与模型。
表 7: a-CTx LvIA/LvD21对
α3β2 nAChRs野生型及其突变型洗脱速率的影响
Figure imgf000063_0001
上表中，
a 毒素肽 α-CTx LvIA/LvD21的浓度； b 阻断后的洗脱时间， 单位为分钟 (min); c 阻断后在洗脱时间内的电流恢复百分数 ( % )； d 95 %置信区间的平均值与误差 （ 6 11±标准误） 。 实施例 5: a-LvIA/LvD21的镇痛活性实验
1. 利用大鼠 CCI模型测定 LvIA/LvD21的镇痛活性
( 1 ) 实验动物和实验材料
利用 SD ( Sprague Dawley )大鼠， 制作坐骨神经慢性挤压伤模型 ( Chronic Constriction Injury model, CCI模型） ， 用压力痛觉测试 仪 (大鼠 800G,型号为美国 IITC 2391) 测定所试芋螺毒素对神经痛的 镇痛活性。 SD ( Sprague Dawley )大鼠， 购自广东省医学实验动物中 心。 CCI 模型的制作参照 Bennett 等（Bennett G J, Xie Y K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man [J]. Pain, 1988， 33(1): 87)的方法。
( 2 ) 实验方法
经腹腔注射戊巴比妥钠 80mg/kg麻醉后，无菌条件下切开右下肢， 暴露坐骨神经主干， 用 4-0铬制羊肠线松扎四道， 间距为 lmm， 结扎 线的松紧以不影响神经外膜的血运为度， 逐层缝合。 左下肢切开暴露 坐骨神经主干但不结扎， 为假手术侧。 各侧伤口缝合前均在局部涂以 青霉素粉剂。 术后腹腔注射青霉素 1次 /天， 8万单位 /次， 连续 3天。 大鼠置管前每笼 5只， 置管后单笼饲养。 将初选合格的大鼠按随机数 字表分成五組， 即生理盐水阴性对照組、 吗啡阳性对照組、 和毒素肽 a-LvIA/LvD21实验組，其中毒素肽 a-LvIA/LvD21实验組重复 2次（即 毒素肽 a-LvIA/LvD21实验組供进行 3次） 。 分别在手术之前、 手术 后三天、 一周、 两周测大鼠患足和假手术侧足的机械痛觉刺激值。 经 检测合格的坐骨神经慢性挤压伤模型 （ CCI ) 模型， 用作测试 LvIA/LvD21对神经痛疗效的整体动物模型。
用腹腔注射给药方式测定 LvIA/LvD21 在 CCI模型上的镇痛作 用。 用生理盐水 (Saline)为空白对照， 也就是阴性对照； 用吗啡 ( Morphine )作阳性对照， 给药剂量为 lmg/kg大鼠体重。 实验組为 毒素肽 a-LvIA/LvD21， 给药剂量为 1 nmol/kg ( ~ 1.7 g/kg ) 大鼠体 重， 每組大鼠数量为 8只（11=8 )。 镇痛活性用 Mechanical Threshold 表示，为观测痛阈值与基础痛阈值（100 )的比值百分数（ % of basal ) , 这个值越大， 镇痛效果越好。
( 3 ) 实验结果
如图 7-8所示。
图 7显示了 a-LvIA/LvD21腹腔给药 （ IP )后 1 - 24小时在 CCI 模型上的镇痛作用。 LvIA/LvD21给药后 1小时就显示出对神经痛的强 镇痛作用， 而阳性对照吗啡在给药后 1 小时却没有镇痛活性； LvIA/LvD21给药后 3小时， 对神经痛的镇痛作用达到最高， 平均镇痛 值为 160 %， 有的高达 200 %， 而阳性对照吗啡在给药后 3小时的平均 镇痛值为 120 % ; 到后 24小时时， LvIA/LvD21的镇痛值仍远高于吗啡 (图 7 ) 。
连续给药 7天后， 测定停药后一周内 （第 7 - 14天）的镇痛值， 结 果如图 8所示。 LvIA/LvD21在 7 - 14天的机械痛觉刺激痛阈值显著分 别高于吗啡組， 在第 12 天时的镇痛效果达到最好， 平均镇痛值达到 200 %， 吗啡組与并且与生理盐水对照組具有显著性差异的镇痛值几乎 没有差异。这说明吗啡停止给药后的镇痛效果就消失了，而 LvIA/LvD21 停止给药后的镇痛效果还得以持续 （图 8 ) ， 表明 LvIA/LvD21不但能 对神经痛起到镇痛作用， 还具有治愈效果。
以上研究结果表明， LvIA/LvD21具有比吗啡更强的镇痛作用， 若 按相同重量剂量计算， 在 CCI模型上， LvIA/LvD21的镇痛效应比吗啡 强 823 - 1176倍。腹腔注射 LvIA/LvD21在大鼠 CCI模型上的镇痛作用 强大且具有很好的持续性， 芋螺毒素自身不会引起成瘾。
2. 利用小鼠热板试验测定 LvIA/LvD21的镇痛活性
( 1 ) 实验动物
实验前将反应潜伏期小于 5s或大于 30s 的小鼠剔除， 选择体重 18±2g的雌性昆明小鼠 50只。 给药前， 将小鼠放在 55±0.5°C的热板测 痛仪（型号为美国 IITC 39 )金属板上， 以小鼠舔后足反应或跳跃反 应的时间计算潜伏期 （S ) 。
( 2 ) 实验方法
依照随机分組数字表分成阴性对照生理盐水（Saline ) 、 阳性对 照吗啡 (Morphine), α-芋螺毒素 LvIA/LvD21共 3組， 每組 10只。 每 組均为侧脑室给药方式， 注射体积为 10 只小鼠。 阳性对照吗啡的 给药剂量为 100 g/kg 小鼠体重； a-LvIA/LvD21 的给药剂量为 0.1 nmol/kg ( ~ 0.17 g/kg ) 小鼠体重。 按同等重量剂量计算， 阳性对照 吗啡的给药量为 LvIA/LvD21 的 588倍。给药前，将小鼠放在 55±0.5。C 的热板测痛仪（型号为美国 IITC 39 )金属板上， 以小鼠舔后足反应 或跳跃反应的潜伏期作为痛阈指标， 单位为秒（s ) 。 每只小鼠测定 2 次取平均值作为基础痛阈， 2次测定时间间隔 5min。 为防足部烫伤， 设 60s为截止时间， 超过 60s者， 痛阈计为 60s。 给药后分别在 15、 30、 45、 60、 90、 120 min时取值作为给药后痛阈， 结果用 Έ±8表示。
( 3 ) 实验结果
如图 9所示。
α-CTx LvIA/LvD21在热板试验模型上显示出很强的镇痛活性（图 9 )。 在给药前 3組小鼠的基础痛阈均在 14 - 17s左右。 给药后， 在所 有时间点， 阴性对照生理盐水（Saline ) 的痛阈仍然维持在 14 - 17s 左右， 在给药后的第 15min， LvIA/LvD21的痛阈迅速增加到 30 s， 吗 啡的痛阈也迅速增加到 32 s (图 9 )， 此时， LvIA/LvD21显示了强大 的镇痛活性， 说明 LvIA/LvD21LvIA的镇痛活性起效非常快。 在给药 后的第 15第 30 - 90min内， LvIA/LvD21的痛阈略微下降持续上升， 而吗啡的痛阈持续下降， LvIA/LvD21 的痛阈与吗啡相比， 痛阈提高 了 1.3 - 1.5倍。在给药后的第 120 min， LvIA/LvD21的痛阈略有下降， 但仍比吗啡给药后此时的痛阈高出 1.3倍。 若按相同重量剂量计算， 在热板模型上， LvIA/LvD21的镇痛效应比吗啡强 764 - 882倍。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人 员将会理解。 根据已经公开的所有教导， 可以对那些细节进行各种修 改和替换， 这些改变均在本发明的保护范围之内。 本发明的全部范围 由所附权利要求及其任何等同物给出。

Claims

1. 一种多肽， 其具有如下的式 I所示的氛基酸序列：
GCCSX XaCX X PX CX 式 I
其中，
表示 D或 H，
X2表示 P、 A或 V，
X3表示 R、 N或 S，
X4表示 N、 V或 A,
X5表示 K、 D、 M或 A，
X6表示 H或 S，
X7表示 D、 E或者 X7缺失，
X8表示 L或 I，
X9表示 G或者 X9缺失。
2. 一种多肽， 其为或者包含选自如下（1)至（3) 中任一项所述 的 ^酸序列：
( 1 ) SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 6、 SEQ ID NO: 11— 15、 SEQ ID NO: 26— 28或 SEQ ID NO: 30中的任一序列 所示的處基酸序列；
( 2 )与上述（ 1 )所述氛基酸序列至少 80%、 优选至少 85%、 更 优选至少 90%、 尤其优选至少 95%、 最优选至少 97%相同的氛基酸 序列； 或
(3)被 1-5个、 优选 1-3个、 更优选 1-2个、 最优选 1个氨 基酸残基的取代、 缺失、 插入和 /或添加而与上述（1)或（2)所示序 列有所不同的處基酸序列。
3. 根据权利要求 1或 2所述的多肽，其中， 所述多肽的 N末端的 第一个半胱氨酸与第三个半胱氨酸形成二硫键， 并且第二个半胱氨酸 与第四个半胱氨酸形成二硫键； 或所述多肽的 N末端的第一个半胱氛 酸与第四个半胱氨酸形成二硫键， 并且第二个半胱氨酸与第三个半胱 氨酸形成二硫键； 或所述多肽的 N末端的第一个半胱氨酸与第二个半 胱氨酸形成二硫键， 并且第三个半胱氨酸与第四个半胱氨酸形成二硫 键； 具体地， 所述多肽的羧基末端是酰胺化的。
4.一种多核苷酸， 其编码权利要求 1至 3中任一项所述多肽的氨 基酸序列。
5.根据权利要求 4所述的多核苷酸，其为或者包含选自如下的（ 1 ) 至（3 ) 中任一项所述的核苷酸序列：
( 1 ) SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 5、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 16 - 21、 SEQ ID NO: 22 - 25、 SEQ ID NO: 29或 SEQ ID NO: 31中的任一序列所示的核苷酸 序列；
( 2 )上面 （1 ) 中所述核苷酸序列的互补序列；
( 3 )在严谊条件下能够与上述（1 ) 中所述的核苷酸序列杂交的 核苷酸序列。
6. 一种核酸构建体， 其包含权利要求 4或 5所述的多核苷酸。
7. 一种表达载体， 其包含权利要求 6所述的核酸构建体。
8. 一种转化的细胞， 其包含权利要求 7所述的表达载体。
9. 一种融合蛋白， 其包含权利要求 1至 3中任一项所述的多肽。
10. 一种药物組合物，其包含权利要求 1至 3中任一项所述的多肽， 或者包含权利要求 9所述的融合蛋白； 可选地，其还包含药学上可接受 的载体或辅料。
11. 一种在体内或体外阻断乙酰胆碱受体或者调节乙酰胆碱水平 的方法， 包括使用有效量的权利要求 1至 3中任一项所述的多肽或权利 要求 9所述的融合蛋白的步骤； 具体地， 所述乙酰胆碱受体是 α3β2乙 酰胆碱受体、 6/ 3 β 2 β 3乙酰胆碱受体或 α3β4乙酰胆碱受体。
12. 一种筛选乙酰胆碱受体抑制剂或者确定乙酰胆碱受体亚型的 方法， 该方法包括： 通过在存在或不存在候选化合物存在的情况下将 乙酰胆碱受体与权利要求 1至 3中任一项所述的多肽或权利要求 9所述 的融合蛋白进行接触的步骤； 具体地， 所述乙酰胆碱受体为 α3β2乙酰 胆碱受体、 6/ 3β2β 3乙酰胆碱受体或 α3β4乙酰胆碱受体。
13. 权利要求 1至 3中任一项所述的多肽或权利要求 9所述的融合 蛋白用于阻断乙酰胆碱受体的用途； 具体地， 所述乙酰胆碱受体是 α3β2乙酰胆碱受体、 （X6/0C3P2P3乙酰胆碱受体或 α3β4乙酰胆碱受 体。
14. 权利要求 1至 3中任一项所述的多肽或权利要求 9所述的融合 蛋白在制备阻断乙酰胆碱受体的药物或试剂中的用途； 具体地， 所述 乙酰胆碱受体是 α3β2乙酰胆碱受体、 （X6/0C3P2P3乙酰胆碱受体或 α3β4乙酰胆碱受体。
15. 权利要求 1至 3中任一项所述的多肽或权利要求 9所述的融合 蛋白在制备治疗和 /或预防和 /或辅助治疗神经系统疾病例如神经痛与 成瘾、 帕金森症、 痴呆、 精神分裂症、 抑郁、 或癌症的药物的用途， 或者用于制备杀灭害虫、 镇痛、 戒烟、 戒毒的药物的用途； 具体地， 所述成瘾由如下原因导致： 各种精神活性物质， 包括尼古丁、 鸦片、 海洛因、 甲基苯丙胺（冰毒） 、 吗啡、 大麻、 可卡因以及国家规定管 制的其它能够使人形成瘾癖的麻醉药品和精神药品等。 具体地， 所述 神经痛由如下原因导致： 癌症与癌症化疗、 酒精中毒、 坐骨神经痛、 糖尿病、 三叉神经痛、 硬化症、 带状疱疹、 机械伤和手术伤、 艾滋病、 头部神经瘫痪、 药物中毒、 工业污染中毒、 淋巴神经痛、 骨髓瘤、 多 点运动神经痛、 慢性先天性感觉神经病、 急性剧烈自发性神经痛、 挤 压神经痛、 脉管炎、 血管炎、 局部缺血、 尿毒症、 儿童胆汁肝脏疾病、 慢性呼吸障碍、 复合神经痛、 多器官衰竭、 脓毒病 /脓血症、 肝炎、 卟 啉症、 维生素缺乏、 慢性肝脏病、 原生胆汁硬化、 高血脂症、 麻疯病、 莱姆关节炎、 感觉神经束膜炎、 或过敏症。
16. 一种治疗和 /或预防和 /或辅助治疗神经系统疾病例如疼痛、烟 酒和毒品成瘾、 智障、 痴呆、 精神分裂症、 中枢神经紊乱、 癫痫症、 帕 金森病、 精神病、 神经肌肉阻滞、 重症肌无力、 抑郁症、 高血压、 心率 不齐、 哮喘、 肌肉松孢、 中风、 乳腺癌和肺癌等的方法， 或者一种杀灭 害虫、 镇痛、 戒烟、 或戒毒的方法， 包括给予有效量的权利要求 1至 3 中任一项所述的多肽或权利要求 9所述的融合蛋白的步骤；具体地，所 述成瘾由烟碱、 吗啡、 可卡因、 酒精等能引起上瘾的物质； 所述神经 痛由如下原因导致： 癌症与癌症化疗、 酒精中毒、 坐骨神经痛、 糖尿 病、 三叉神经痛、 硬化症、 带状疱疹、 机械伤和手术伤、 艾滋病、 头 部神经瘫痪、 药物中毒、 工业污染中毒、 淋巴神经痛、 骨髓瘤、 多点 运动神经痛、 慢性先天性感觉神经病、 急性剧烈自发性神经痛、 挤压 神经痛、 脉管炎、 血管炎、 局部缺血、 尿毒症、 儿童胆汁肝脏疾病、 慢性呼吸障碍、 复合神经痛、 多器官衰竭、 脓毒病 /脓血症、 肝炎、 卟 啉症、 维生素缺乏、 慢性肝脏病、 原生胆汁硬化、 高血脂症、 麻疯病、 莱姆关节炎、 感觉神经束膜炎、 过敏症。
17. 权利要求 1至 3中任一项所述的多肽的制备方法， 包括下述步 1 )在 ABI Prism 433a 多肽合成仪上或者手工方法合成线性多肽， Fmoc ^酸的侧链保护基为： Pmc(Arg)、 Trt或 Acm(Cys)、 But (Thr、 Ser、 Tyr)、 OBut (Asp)以及 Boc (Lys);
2)将步骤 1) 中合成的线性多肽从树脂上切割下来；
3)用冰乙醚沉淀和洗涤步骤 2) 中得到的线性多肽， 回收得到线 性多肽粗品；
4)用制备型反向 HPLC C18 柱（Vydac) 纯化步骤 3 ) 中得到的 线性多肽粗品；
5)将步骤 4) 中得到的产物进行两步或一步氧化折叠。
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