WO2013185572A1 - αO-超家族芋螺毒素肽、其药物组合物及用途 - Google Patents

αO-超家族芋螺毒素肽、其药物组合物及用途 Download PDF

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WO2013185572A1
WO2013185572A1 PCT/CN2013/076967 CN2013076967W WO2013185572A1 WO 2013185572 A1 WO2013185572 A1 WO 2013185572A1 CN 2013076967 W CN2013076967 W CN 2013076967W WO 2013185572 A1 WO2013185572 A1 WO 2013185572A1
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polypeptide
neuralgia
receptor
cysteine
blocking
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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
J.迈克尔 麦金托什
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Hainan University
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Hainan University
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Priority to JP2015516419A priority Critical patent/JP6324952B2/ja
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Priority to US14/407,770 priority patent/US9718864B2/en
Publication of WO2013185572A1 publication Critical patent/WO2013185572A1/zh
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Definitions

  • the invention belongs to the field of biochemistry and molecular biology, and relates to an oc O-superfamily 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 nicotinic acetylcholine receptors (nAChRs) or NMDA receptors (NMDAR), and pharmaceutical use of the conotoxin peptides.
  • nAChRs nicotinic acetylcholine receptors
  • NDA receptors NMDA receptors
  • Nicotinic acetylcholine receptors are cell membrane proteins that are ubiquitous in the animal kingdom and have important physiological and clinical significance. They mediate the physiological functions of many central and peripheral nervous systems, including learning, memory, response, analgesia and exercise. Control, etc. nAChRs activate dry release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and Y-acetylbutyric acid. nAChRs have been shown to be key targets for screening and treating a wide range of important disease drugs, including addiction, pain, cancer, mental retardation, Parkinson's disease, psychosis, depression, myasthenia gravis and other intractable diseases.
  • nAChR agonists such as nicotine
  • nAChRs have strong side effects on the heart and gastrointestinal tract and are addictive. Therefore, the development of ligand drugs with high selectivity for various subtypes of nAChRs is the key to the treatment of these diseases ( Livett BG, Sandall DW, Keays D, Down J, Gayler KR, Satkunanathan N, Khalil Z. Therapeutic applications of conotoxins To target, the neuronal nicotinic acetylcholine receptor. Toxicon. 2006, 48(7): 810-829. Nicke, A., Wonnacott, S.
  • the premise of developing such a drug is to obtain a selective compound that can specifically bind to various subtypes of nAChRs, or directly as a therapeutic drug for a related disease, or as a tool to study and identify the fine composition of various subtypes. And physiological functions.
  • tissue such as breast cancer and lung cancer
  • activation of nicotinic acetylcholine receptors on tumor cell membranes 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 (X and p subunits into a variety of subtypes, each with distinct pharmacological characteristics. Due to the lack of highly selective ligand compounds for various subtypes, research and clarification of each The fine structure and function of the various subtypes of nAChRs faces many challenges.
  • oc 9 oc 10 nAChR blocker has the function of treating neuralgia and accelerating the recovery of injured nerves. , may be through immune mechanisms (Holtman, JR; Dwoskin, LP; Dowell, C; Wala, EP; Zhang, ⁇ ; Crooks, PA; Mcintosh, JM, The novel small molecule alpha9alpha10 nicotinic acetylcholine receptor antagonist ZZ-204G Is analgesic. European journal of pharmacology 2011, 670 (2-3), 500-8.
  • the oc 9 subunit variant affects the transformation and proliferation of bronchial cells, which is of great importance in the treatment of lung cancer (Chikova, A.; Grando, SA, Naturally occurring variants of human Alpha9 nicotinic receptor differentially affect bronchial cell proliferation And transformation. PloS one 2011, 6 (11), e27978. ).
  • the pain affects 1/6 of the population, including arthritis, neuralgia, swelling and pain.
  • neuralgia affects 4-8% of the population.
  • Existing methods of treating neuralgia mainly local anesthetics, block pain signals due to peripheral nerves, plexus, dorsal root nerves, sympathetic nervous system, and the like.
  • these treatments can only have an analgesic effect for a short time, but they cannot cure the neuralgia.
  • 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, osteosarcoma, 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.
  • Neuropathic treatments targeting a 9 a 10 nAChR can exert analgesic effects by intramuscular injection (Vincler, M. Wittenauer, S. Parker, R. Ellison, M. Olivera, BM Mcintosh, JM Molecular mechanism for analgesia involving Specific antagonism of alpha9alphal0 nicotinic acetylcholine receptors. Proc Natl Acad Sci USA, 2006,103 ( 47 ) : 17880-4 ⁇ ), is easier than the current commercial ⁇ -CTX MVIIA analgesic drug-picoginopeptide administration route, Chico norpeptide needs to be directly administered to the spine by a programmed pump built into the human body.
  • nAChRs nicotinic acetylcholine receptors
  • a variety of nAChRs subtypes are not only a drug target for nicotine addiction, but also a drug target for drug addiction such as morphine and cocaine.
  • the NMD receptor (N-methyl-D-aspartate receptor) is an important excitatory acid receptor in the central nervous system. It is a ligand-gated ion channel receptor with extensive physiological and pharmacological significance. . NMDA receptors not only play important physiological roles in the development of the nervous system, such as regulating neuronal survival, regulating neuronal dendrites, axon structure development and involvement in synaptic plasticity formation; The NMDA receptor also plays a key role. At the ischemic concentration site, NMDA receptors are activated, causing extracellular calcium influx, leading to cell death (Twede, VD, Miljanich, G., Olivera, BM & Bulaj, G.
  • NMDA receptors are mainly distributed in the central system, such as the brain and spinal cord; NMDA receptors are also distributed in the periphery, such as NR3B is mainly expressed in motor neurons, while peripheral NMDA receptors are in facial muscle pain. And plays an important role in the formation of edema.
  • NMDA receptors are a vital receptor in the process of learning, memory, pain, etc., and are also targets for the onset and treatment of many neurological diseases, including intractable pain. Pain, drug and alcohol addiction, epilepsy, ischemia, Parkinson's disease, dementia, excitatory neuronal cell death, etc. (Sattler, R. et al. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD -95 protein. Science (New York, NY) 1999, 284, 1845-1848. Lewis, RJ & Garcia,
  • toxins conopeptide, conotoxin, CTX
  • toxins produced in the carnivorous mollusks of the carnivorous mollusks living in the tropical sea have attracted much attention and have been used to systematically study and develop the specificity of various subtypes of nAChRs. Blocker.
  • Conopeptide (conotoxin, CTX) is a cysteine-rich (Cys)-rich neuropeptide toxin composed of 7-50 amino acid residues. Conotoxin is classified into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence of its precursor protein and the cysteine pattern. Up to now, all known conotoxins can be divided into 18 superfamilies. A, B, C, D, S, M, II, 12, 13, J, L, 01, 02, 03, P, T, respectively V, Y ( Kaas Q, Yu R, Jin AH, Dutertre S, Craik DJ (2012) ConoServer: updated content, knowledge, and discovery tools in the conopeptide database.
  • the a-type conotoxin (oc*-CTX) has the function of blocking nicotinic acetylcholine receptors (nAChRs); the conotoxin peptide Conantokins which does not contain cysteine has a blocking N-methyl-D-day
  • NMDAR nicotinic acetylcholine receptor
  • Each superfamily conotoxin can be divided into oc, oc A, ⁇ ⁇ ( ⁇ -superfamily), ⁇ , ⁇ , ⁇ , ⁇ ⁇ (0-superfamily), ⁇ , ⁇ , depending on the type of receptor target.
  • Conotoxin has a special function that specifically binds to various ion channels in animals. At present, conotoxin has attracted much attention and has been used to systematically research and develop specific blockers of various subtypes of nAChRs. Summary of the invention
  • the inventors discovered a new class of oc O-superfamily conotoxin peptides through intensive research and creative labor.
  • the present inventors have surprisingly found that the oc O-superfamily conotoxin peptide of the present invention is capable of specifically blocking acetylcholine receptors and NMDA receptors, particularly for neuropathic drug targets, breast cancer, lung cancer target oc 9 (x l0 nAChR has the strongest blocking activity, and has a good application prospect in the preparation of analgesic drugs, anti-addiction, epilepsy, or cancer drugs, as well as neuroscience tools and the like.
  • One aspect of the invention relates to a polypeptide which is or comprises one or more of the same or different amino acid sequences of any one of (1) to (3) selected from: (1) an amino acid sequence as shown in any one of SEQ ID NOS: 7-12;
  • 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;
  • SEQ ID NO: 7 or 8 is a wild type precursor peptide designated GeXIVAWT;
  • SEQ ID NO: 9 is a mature peptide thereof.
  • SEQ ID NO: 10 or 11 is a mutant precursor peptide designated GeXIVA; SEQ ID NO: 12 is its mature peptide.
  • the precursor peptide of GeXIVA and its wild-type GeXIVAWT contains three regions of signal peptide, propeptide and mature peptide, and their specific sequences and their analysis are as described in Examples 1-2.
  • the wild-type mature peptide has five cysteines, which are different from the even-numbered cysteine patterns of all conotoxins previously found. It is an ocO-superfamily new conotoxin, which blocks oc9 lO nAChR It is the most active, so it is named ocO-superfamily conotoxin (a O-conotoxin).
  • 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 column of homologous polypeptides differs from any of SEQ ID NOS: 1-4 in that it may be substituted, inserted, added and/or deleted by one or more, preferably 1-5, more preferably 1- Three, particularly preferably one to two, most preferably one, an amino acid residue.
  • the amino acid change is a minor change in properties, ie a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; small fragment deletions, usually from 1 to about a deletion of 5, preferably 1-3, more preferably 1 amino acid; a small aryl or carboxy terminal extension, such as a methionine residue added at the amino terminus; a small amount of up to about 20-25 residues Linking the peptide; or facilitating purification of small extensions such as polyhistidine fragments, epitopes or binding regions by altering the net charge or other function.
  • small fragment deletions usually from 1 to about a deletion of 5, preferably 1-3, more preferably 1 amino acid
  • a small aryl or carboxy terminal extension such as a methionine residue added at the amino terminus
  • a small amount of up to about 20-25 residues Linking the peptide or facilitating purification of small extensions such as polyhistidine fragments, epitopes or binding regions by altering the net charge or other function.
  • examples of conservative substitutions are in basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), and polar amino acids. (Glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), similarly shaped amino acids (between arginine and serine), and cysteine Amino acids (cysteine, arginine, serine, tryptophan, glycine, tyrosine acid (glycine, alanine, serine, threonine, and methionine) with one base difference in codons Substitutions carried out.
  • Amino acid substitutions which generally do not alter 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, Ala/Val, Ser/Gly, Arg/Ser, Cys/Ser, Arg/Cys, Arg/Ala, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, etc. and reverse substitutions.
  • 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 oc O-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 one of the present invention wherein the first cysteine at the N-terminus in SEQ ID NO: 12 forms a disulfide bond with the second cysteine, and the third cysteine
  • the acid forms a disulfide bond with the fourth cysteine; or the first cysteine forms a disulfide bond with the third cysteine, and the second cysteine and the fourth cysteine
  • the acid forms a disulfide bond; or the first cysteine forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine;
  • the carboxy terminus of the polypeptide is a free C-terminus or is amidated.
  • the above polypeptide of the present invention is a conotoxin peptide; specifically, it is ( ⁇ -conotoxin peptide.
  • the above conotoxin peptide can be extracted from ins C. generalis produced in Hainan, China, or The gene is deduced to obtain a mature peptide sequence.
  • the amino acid sequence can also be chemically synthesized (for example, the method in Reference Example 3); or the nucleotide can be expressed by means of genetic recombination (Preparation of nucleotide sequence) -2 or directly synthetic) to obtain the polypeptide. 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:
  • the side chain protecting groups of Fmoc® acid are: Pmc(Arg), Trt(Cys), But (Thr, Ser, Tyr), OBut (Asp) , Boc (Lys); cysteine with a Trt or Acm protecting group, respectively forming a disulfide bond between the corresponding cysteines; or all cysteines with a Trt protecting group, by one-step method The oxidative fold randomly forms a disulfide bond.
  • step 2) The product obtained in step 2) is subjected to two-step or one-step oxidative folding.
  • Another aspect of the invention relates to a polynucleotide encoding a cyanoic acid sequence of a polypeptide of any of the invention.
  • nucleotide sequence of any one of (1) to (3) is selected from one or more of the following:
  • SEQ ID NOS: 1 - 6 are the coding sequences of SEQ ID NOS: 7-12, respectively.
  • SEQ ID NO: 1 or 2 encodes a wild-type precursor peptide designated GeXIVAWT;
  • SEQ ID NO: 3 encodes a mature peptide thereof.
  • SEQ ID NO: 4 or 5 encodes a mutant precursor peptide designated GeXIVA;
  • SEQ ID NO: 6 encodes its mature peptide.
  • hybridization between polynucleotides numerous references are available in the prior art, including, for example, Sambrook et al., Molecular Cloning Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989.
  • Various degrees of stringent conditions can be applied in hybridization, such as moderate, moderate-high, or highly stringent conditions. The more stringent the conditions, the higher the complementarity required to form a double helix.
  • the degree of rigor can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like.
  • a further aspect of the invention relates to a primer pair which is or comprises the nucleotide sequence set forth in SEQ ID NO: 13 and SEQ ID NO: 14. The primer pair can be used for PCR amplification to obtain the polynucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • 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 a recombinant expression vector comprising the nucleic acid construct of the invention; in particular, the recombinant vector is a recombinant pET22b(+) vector.
  • a further aspect of the invention relates to a transformed cell comprising a recombinant expression vector of the invention; in particular, the transformed cell is a transformed Sf9 cell.
  • 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; Accepted carrier or excipient.
  • a further aspect of the invention relates to a method of blocking an acetylcholine receptor and an NMDA receptor, comprising the step of using an effective amount of the polypeptide or fusion protein of any of the invention; in particular, the acetylcholine receptor is (X 9 (X 10 acetylcholine receptor, or other acetylcholine receptor subtype and NMDA receptor).
  • a further aspect of the invention relates to a method of screening for an acetylcholine receptor inhibitor and an NMDA receptor or determining an acetylcholine receptor and an NMDA receptor subtype, the method comprising: by presenting in the presence and absence of a candidate compound The step of contacting the acetylcholine receptor or the NMDA receptor subtype with the polypeptide or fusion protein of any of the present invention; specifically, the acetylcholine receptor is (X 9 (X 10 acetylcholine receptor; or the NMDA) The receptor is NR2C NMDA receptor.
  • oc 0-conotoxin GeXIVA can specifically block the a 9 a 10 acetylcholine receptor at a very low concentration, it is concluded that the acetylcholine receptor is a 9 a 10 subtype of acetylcholine receptor.
  • the oc O-conotoxin GeXIVA is capable of specifically blocking the NR2C NMDA receptor at a lower concentration, the NMDA receptor is inferred to be the NR2C ⁇ DA receptor.
  • a further aspect of the invention relates to any of the inventions Use of the polypeptide or fusion protein described for blocking the acetylcholine receptor and the NMDA receptor; specifically, the acetylcholine receptor is (X 9 (X 10 acetylcholine receptor, or other B) Cholinergic receptor subtypes; NR2C NMDA receptors and other subtypes.
  • a further aspect of the invention relates to the use of a polypeptide or fusion protein according to any of the invention for the preparation of a medicament or agent for blocking an acetylcholine receptor and an NMDA receptor; in particular, the acetylcholine receptor is a 9 10 Acetylcholine receptor, with NR2C NMDA receptor.
  • a further aspect of the invention relates to the preparation of a polypeptide or fusion protein according to any of the invention for the treatment or prevention of diseases of the nervous system such as neuralgia, breast cancer, lung cancer, addiction, epilepsy, ischemia, Parkinson's disease a drug such as dementia, or excitatory neuronal cell death, or a drug for preparing a drug for killing a pest (for example, Lepidoptera: Noctuidae), analgesic, smoking cessation, or detoxification; specifically, the nerve
  • the pain is caused by the following reasons: 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, osteosarcoma, multiple Point motor neuralgia, chronic congenital sensory neuropathy, acute strenuous spontaneous neuralgia, crush neuralgia, vasculitis,
  • a further aspect of the invention relates to a method of treating and/or preventing and/or adjuvant treatment of a nervous system disorder such as neuralgia, breast cancer, lung cancer, addiction, epilepsy, ischemia, Parkinson's disease, dementia, or excitement
  • a nervous system disorder such as neuralgia, breast cancer, lung cancer, addiction, epilepsy, ischemia, Parkinson's disease, dementia, or excitement
  • the neuralgia is caused by: cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis, Herpes zoster, mechanical
  • the conotoxin peptide of the present invention can exert analgesic activity by binding (X 9 (X 10 acetylcholine receptor (nAChR) or/and NR2C NMDA receptor. It can be used for research, diagnosis and treatment of neuralgia, breast cancer , lung cancer, Parkinson's disease, dementia, addiction, eclampsia, ischemia and other neurological diseases, as well as useful molecular probes for research, etc. Different (X-type CTX to vertebral pusher receptors) Affinity is different, sometimes differs by several orders of magnitude.
  • X-type CTX can be used as a useful probe to study the phylogeny of spinal promoter nAChR, which can be used as a molecular probe to determine the difference in nAchR Asian Type. They are drug candidates, lead drugs, and therapeutic drugs for new drug development. An explanation of the terms involved in the present invention is given below.
  • 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, osteoma, multi-point motor neuralgia, chronic congenital sensory neuropathy, acute strenuous spontaneous neuralgia, crush 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.
  • neuralgia including cancer and cancer chemotherapy, alcoholism, sciatica, diabetes, trigeminal neuralgia, sclerosis,
  • 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 Express it.
  • 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-chain nucleic acid molecules that are isolated from a native gene, or modified to contain nucleic acid fragments that are combined and juxtaposed in a non-natural manner.
  • nucleic acid construct is synonymous with an expression cassette when the nucleic acid construct comprises all of the regulatory sequences necessary for expression of a coding sequence of the invention.
  • coding sequence is defined herein as the portion of a nucleic acid sequence that directly determines the column of its protein product.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • An isolated nucleic acid sequence encoding a peptide of the 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 as used herein 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. Such regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequence should include a promoter and a termination signal for transcription and translation. In order to introduce a specific limit for the regulation of the joint with the joint ⁇ .
  • operably linked is defined herein as two conformations 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 end of the polypeptide, which directs the encoding of the polypeptide into the cell's secretory pathway.
  • the 5th end of the coding region of the nucleic acid sequence may naturally contain a translational reading frame consistent with the coding region of the secreted polypeptide.
  • Linked signal peptide 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. However, any can be directed to the present 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.
  • a propolypeptide is generally inactive and can be converted to a mature active polypeptide by cleavage of the propeptide from the propolypeptide by catalytic or autocatalytic.
  • the peptide region is immediately adjacent to the ⁇ terminus 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 a regulatory compound, to open or shut down gene expression.
  • 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 nucleotide 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 (ie, a complete structure existing outside the chromosome, which may be Replica on a chromosome, such as a plasmid, an extrachromosomal element, a tiny chromosome, 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, that will generally be introduced into the entire DNA of the host cell genome.
  • 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 mutated such that it becomes 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.
  • the increase in copy number of the core can be accomplished by inserting at least one additional copy of the sequence into the host cell genome, or by inserting an amplifiable selectable marker with the nucleic acid sequence, by culturing the cell in the presence of a suitable selection reagent , selecting a cell containing an amplified copy of the selectable marker gene, thereby containing an additional copy of the nucleus.
  • the invention also relates to a recombinant host cell comprising a nucleic acid sequence of the invention that can be used to recombinantly produce a polypeptide.
  • a vector comprising a nucleic acid sequence of the present invention can be introduced into a host cell such that the vector is maintained in the form of the above-described chromosomal integrant or self-replicating extra-chromosomal vector.
  • host cell covers any parent and parent due to mutations that occur during replication. Different progeny of cells. 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.
  • 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 provided 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 the cell lysate.
  • 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
  • Control of pests can be accomplished using a conotoxin peptide or polynucleotide of the invention by a variety of methods known to those skilled in the art. These methods include, for example, the recombinant microorganism should For use in pests (or their loci), and transformation of plants with a gene encoding a conotoxin peptide of the 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 as a root treatment or as a complete plant treatment in the late 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 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/mg. These formulations will be used in an amount of about 50 mg (liquid or dry) per hectare of one lkg or more.
  • 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
  • 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 with neuralgia, breast cancer, lung cancer, mental retardation, addiction, pain, Parkinson's disease, psychosis, depression, myasthenia gravis, eclampsia, ischemia, etc.
  • the disease or condition involved 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, delivery site, method of administration, administration of the individual patient. The schedule and other factors known to the doctor. Therefore, the "effective amount" for the purposes of this paper is determined by these considerations.
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the present invention is administered parenterally, orally, intracisternally, intrathecally, and the like.
  • “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid Or liquid fillers, diluents, capsules or any type of formulation aid.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intrathecal, and intraarticular injections and infusions.
  • the polypeptides of the invention may also be administered as appropriate by a slow-drying system.
  • the invention also relates to pharmaceutical compositions that specifically block nAChRs or / and NMDA receptors.
  • the conotoxin peptide of the present invention can be used as a useful probe for studying the phylogeny of animal nAChRs or/and NMDARs; as a molecular probe to determine different subtypes of nAChRs or/and NMDARs; as a molecular model, design New drug; as a tool for the diagnosis and diagnosis of neurological diseases such as Parkinson's disease, mobility disorder, schizophrenia, epilepsy, ischemia, etc.; treatment of neuralgia, addiction, breast cancer, lung cancer, small Candidate drugs for cell lung cancer, epilepsy, ischemia, etc.
  • a peptide insecticide it has been developed as a new type of biological pesticide.
  • the a 0-conotoxin peptide of the present invention is capable of specifically blocking acetylcholine receptors ( nAChRs ) and NMDA receptors, and has analgesic activity and inhibits the growth of breast cancer and lung cancer cells, and treats addiction and epilepsy. Efficacy with ischemia and the like. DRAWINGS
  • nAChRs and NMDA receptors are not indicated in the figure, they are the corresponding receptors in rats.
  • the source of receptor type in rats is omitted from the legend and figure.
  • Figure 1 shows the oc O-GeXIVA mature peptide sequence (SEQ ID NO: 1) and the isomers with three possible disulfide linkages.
  • the disulfide bond of GeXIVA12 is 1-11, III-IV; the double bond of GeXIVA13 is 1-111, II-IV; the disulfide bond of GeXIVA14 is I-IV, 11-111.
  • FIG. 2 A shows the effect of 33 nM a O GeXIVA12 on the current of oc 9 oc 10 nAChR.
  • C refers to the control current
  • the arrow refers to the current trace formed by the first Ach pulse after 5 minutes of incubation with 33 nM ⁇ O GeXIVA12 ( ⁇ 0 nA ).
  • the Ach pulse time is Is;
  • the coordinates are the current magnitude, the unit is nA, and the abscissa is the cumulative Time, the unit is ms, and the interval between each adjacent two current tracks before and after the incubation is 60s.
  • B, C, and D are the three isomers a 0-GeXIVA12, a 0-GeXIVA13, and ocO-GeXIVA 14 pairs (x9 (xl0nAChR concentration dose response curve.
  • the abscissa in Figure B, C, D is the used ocO).
  • the logarithm of the molar concentration (M) of the GeXIVA isomer (Log [Toxin Concentration] M ); the ordinate is the dose response percentage (% Response), which is the ratio of the acetylcholine receptor current to the control current at the corresponding concentration of the toxin. Percentage, the percentage of response per dose is the mean (mean) of the data recorded for 6-12 Xenopus oocytes, and the curve also shows the standard error (SEM).
  • FIG. 3 Concentration dose response curve (AE) of oc O GeXIVA12 to other nAChRs subtypes.
  • the abscissa is the logarithm of the molar concentration (M) of ocO-GeXIVA12 used (Log[Toxin Concentration] M ); the ordinate is the percentage of dose response (% Response), which is the acetylcholine receptor current under the corresponding concentration of toxin. Percentage of control current, the percentage of response per dose is the mean (mean) of the data recorded by 6-12 African claw pond oocytes, and the curve also shows the standard error (SEM).
  • SEM standard error
  • nAChRs subtype and the half-blocking dose (IC 5 .) for this subtype are indicated on the map.
  • a O GeXIVA12 has a large blocking activity on oc*p4 and oc*p2 nAChRs, and the blocking activity against a* ⁇ 2 nAChRs is much higher than that on oc * ⁇ 4 nAChRs; on mouse muscle type nAChR ( ⁇ ⁇ )
  • the blocking activity is similar to that of the oc7 nAChR subtype.
  • Figure 4 shows the concentration-dose response curves of three isomers ocO-GeXIVA12, GeXIVA13 and GeXIVA14 to OC90C10 nAChR with cesium ion ND96 perfusate (Ba ++ -ND96 ) instead of conventional ND96 perfusate.
  • the coordinates are the logarithm of the molar concentration (M) of the three oc O-GeXIVA isomers used (Log[Toxin Concentration] M ); the ordinate is the dose response percentage (% Response), which is the acetylcholine exposure under the corresponding concentration of toxin The ratio of the ratio of the body current to the control current.
  • the percentage of response per dose was the mean (mean) recorded for 9 Xenopus oocytes, and the curve also showed standard error (SEM). The corresponding isomers are indicated for the half-blocking dose of the oc 9 oc 10 nAChR subtype (IC 5 .).
  • FIG. 5 Concentration dose response of various subtypes of NMDA receptor by oc O GeXIVA12 Line.
  • the abscissa is the logarithm of the molar concentration (M) of oc O-GeXIVA12 used; the ordinate is the percentage of dose response (% Response), which is the ratio of the ratio of NMDA receptor current to the control current for the corresponding concentration of toxin.
  • the percentage of dose response was the mean (mean) recorded for 3-5 African claw pond oocytes, and the curve also showed the standard error of 95% confidence interval (SEM).
  • SEM standard error of 95% confidence interval
  • FIG. 6 Concentration dose response curves of the isoforms 0-GeXIVA12, O GeXIVA13 and O GeXIVA14 to various other subtypes of the nAChR receptor (A-J). The various meanings in the figure are the same as in Figure 3.
  • FIG. 7 Concentration dose response curves (A-F) of three isoforms a O GeXIVA12, a O GeXIVA13 and a O GeXIVA14 on human nAChR receptor-associated subtypes.
  • the various indications in the figure have the same meaning as in Figure 3.
  • FIG. 8 Concentration dose response curves (AK) of three isoforms a O GeXIVA12, a O GeXIVA13 and a 0-GeXIVA14 against rat oc 9 (X 10 nAChR receptors). The meaning is the same as in Figure 3.
  • Figure 9 shows oc O-GeXIVA12 blockade (x 9 (x l0 nAChR, can not prevent the blocking effect of oc -CTX RgIA [S4T; R9Cit; YlOIodo, R11Q] on oc 9 oc 10 nAChR, proves both The site of binding to oc 9 oc 10 nAChR is completely different.
  • Figure 10 shows the inhibitory effect of recombinant a O-superfamily conotoxin wild type GeXIVAWT (rCTX-K41) on Sf9 cells.
  • the abscissa is the concentration of recombinant GeXIVAWT in g/ml
  • Control is the negative control without recombinant GeXIVAWT
  • the ordinate is at 490 nm measured on the enzyme-labeled immunoassay instrument (BIO-RAD MODEL 550) Absorbance values.
  • the C. generalis living body collected from the coastal areas such as Hainan Island and Xisha Islands is used as a material.
  • a small column of centrifugal tissue/cell total RNA extraction kit (Shanghai Huasheng Biotechnology Co., Ltd.) was used to extract total RNA according to the manual, and then cDNA synthesis was performed. Specific steps can be found in the literature (eg, Quan Yaru, Ru Sulan, Lin Qiujin, Chang Sun Dongting, Zhang Ben. Extraction of conotoxin RNA and cDNA synthesis, Chinese Marine Medicine, 2005, 24 ( 2 ) :1-5 )get on.
  • primers were designed based on the untranslated region sequence of the 01-gene superfamily precursor gene, and subjected to RT-PCR amplification to obtain a specific PCR amplification product.
  • the primer sequences used are:
  • Primer 1 5, -CATCGTCAAGATGAAACTGACGTG-3' (SEQ ID NO: 13);
  • the RT-PCR cycle procedure was: 94 pre-denaturation for 3 minutes, 94 denaturation for 30 seconds, 56 annealing for 30 seconds, 72 extension for 30 seconds, 30 cycles, and 72 extensions for 2 minutes.
  • the above specific PCR product was recovered, and ligated with T-easy vector (Promega) to transform Escherichia coli XL1 strain (other commercial competent E. coli cells can also be used), and blue and white colonies and ampicillin resistance were used to select recombinants. Purify the recombinant plasmid for sequencing, and select different clones (for example, 3 - 5 different clones) from the same PCR product. Sequencing, analysis.
  • the cDNA gene of the wild-type precursor peptide GeXIVAWT which is a novel member of the 01-superfamily conotoxin of the present invention, was obtained.
  • the GeXIVAWT precursor peptide gene was analyzed by DNAStar software, and its open reading frame (ORF) sequences, SEQ ID NO: 1 and SEQ ID NO: 2, were obtained as follows:
  • An open reading frame (ORF) encoding the GeXIVAWT precursor protein (existing allelic variation, the 19th position of the bordered T and G indicates its single base mutation site; the underlined part indicates the DNA sequence encoding the mature peptide):
  • the amino acid sequence of the above-mentioned sequence encoding the new member of the Ol-superfamily conotoxin, the wild-type precursor peptide GeXIVAWT (hereinafter also referred to as a O-conotoxin GeXIVAWT precursor or ⁇ -GeXIVAWT precursor or GeXIVAWT precursor) is as follows ( The 7th position L and V of the framed frame indicate the amino acid of the mutation site, the underlined part indicates the amino acid sequence of the signal peptide, and the arrow indicates the post-translational modification processing site of the mature peptide.
  • LPK i TCRSSGRY CRSPYDCRRRYCRRITDACV ( SEQ ID NO: 7 ); or mKLTC ⁇ YI ⁇ l ⁇ l, TACO1, TTAVTYSRGEHKHRALMSTGTNY RLPK i TCRSSGRY CRSPYDCRRRYCRRITDACV (SEQ ID NO: 8).
  • the wild-type GeXIVAWT precursor peptide cDNA gene encodes a signal peptide and a mature peptide via the ItP line of ProP 1.0 Server (Duckert, P.; Brunak, S.; Blom,
  • the nucleotide sequence encoding the GeXIVAWT mature peptide is as follows (the border portion indicates the coding half)
  • TATAG SEQ ID NO: 3
  • the cyano acid sequence of the GeXIVAWT wild-type mature peptide (hereinafter also referred to as oc O-conotoxin GeXIVAWT or oc O-GeXIVAWT or GeXIVAWT) is as follows (the cysteine is added to the border portion):
  • T@RSSGRY@RSPYD@RRRY@RRITDA@V ( SEQ ID NO: 9 ) represents The wild type GeXIVAWT precursor peptide contains three regions of signal peptide, propeptide and mature peptide, and the seventh amino acid residue in the signal peptide. Is leucine or valine (L or V), the corresponding codon is TTG or GTG.
  • the wild-type mature peptide region (SEQ ID NO: 9) contains 5 cysteine acids (Cys), which was found in the past.
  • the conotoxins are different, as shown in Table 1 for comparison with members of the related gene superfamily.
  • Example 2 Preparation and sequence analysis of a novel a 0-superfamily conotoxin gene mutant (GeXIVA)
  • the 181 - 183 TGC of the wild-type GeXIVA WT precursor peptide gene encodes a cysteine Cys, which is point-mutated (also obtained by direct artificial chemical synthesis of SEQ ID NO: 4), ie, a single base mutation to a coding
  • the codon CGC (@GC ⁇ GC) of the Arg (R), ie, the T at position 181 becomes C (T181C), and the point mutant is named GeXIVA precursor peptide gene, and its sequence is as follows:
  • An open reading frame (ORF) encoding the GeXIVA precursor protein (there is an allelic variation, the 19th position of the bordered T and G indicates its single base mutation site; the underlined part indicates the 181th mutation site, underlined Partially indicates the DNA sequence encoding the mature peptide):
  • the above sequence encodes a new member of the Ol-superfamily conotoxin, the mutant precursor peptide GeXIVA (hereinafter also referred to as oc O-conotoxin GeXIVA precursor or oc O-GeXIVA precursor or GeXIVA precursor).
  • the 7th position L and V represent the amino acid of the mutation site
  • the double underlined part indicates the artificial mutation site at position 61
  • the underlined part indicates the amino acid sequence of the signal peptide
  • the arrow indicates the post-translational modification processing position of the mature peptide. Point, italic indicates the N-terminal propeptide region);
  • LPK i TCRSSGRY CRSPYDRRRRYCRRITDACV (SEQ ID NO: 10).
  • the signal peptide and mature peptide encoded by the mutant GeXIVA precursor peptide cDNA gene were tested by online ProP 1.0 Server (Duckert, P.; Brunak, S.; Blom, ⁇ , Prediction of proprotein convertase cleavage sites. Protein Engineering, design & selection: PEDS 2004, 77 (1), 107-12 ⁇ ).
  • the nucleotide sequence encoding the GeXIVA mature peptide is as follows (the bordered portion indicates the codon that encodes cysteine; the double-underlined letter C indicates the single base mutation corresponding to the point mutation amino acid
  • TATAG SEQ ID NO: 6
  • the amino acid sequence of the GeXIVA mutant mature peptide (hereinafter also referred to as a O-conotoxin GeXIVA or a O-GeXIVA or GeXIVA) is as follows (the border portion is cysteine, and the double-underlined letter R indicates a point mutation base) Acid):
  • the mutant GeXIVA precursor peptide contains three regions of a signal peptide, a propeptide and a mature peptide, and the amino acid residue at the 7th position in the signal peptide is leucine or valine (L or V), and the corresponding codon is TTG or GTG.
  • the mutant mature peptide region contains four cysteines (Cys), which are different from the previously found conotoxin, and are shown in Table 1 above for members of the related gene superfamily.
  • GeXIVA is a blocker of nAChRs and NMDARs and has the strongest blocking activity against a 9 a 10 nAChR, so it is officially named oc O-conotoxin GeXIVA, abbreviated as oc O- GeXIVA or GeXIVA.
  • Example 3 Synthesis of ⁇ -conotoxin GeXIVA
  • the resin peptides of the three isomers were artificially synthesized by Fmoc chemistry, except for cysteine, and the remaining amino acids were protected with standard side chain protecting groups.
  • the 3rd and 4th cysteine (Cys) of GeXIVA12 are protected by Trt (S-trityl), and the 1st and 2nd cysteine-SH are protected by Acm (S-acetamidomethyl) ;
  • GeXIVA13 The 2nd and 4th cysteine (Cy)-SH are protected by Trt (S-trityl), and the 1st and 3rd cysteine-SH are protected by Acm (S-acetamidomethyl) in pairs;
  • the 2nd and 3rd cysteine (Cys) of GeXIVA14 are protected by Trt (S-trityl), and the 1st and 4th cysteine-SH are protected by Acm (S-acetamidomethyl).
  • the synthesis steps were 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 synthesis step reference instrument synthesis manual get on. In order to complete the reaction, the piperidine deprotection and coupling time were appropriately extended, and the refractory acid was double-coupled to obtain a resin peptide.
  • 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
  • the crude linear peptide was recovered and purified by preparative reverse-HPLC C18 column (Vydac) with a linear gradient of 10-50% B60 in 0-40 min.
  • Solvent B was 60% ACN (acetonitrile), 40% H20, 0.92% TFA (trifluoroacetic acid
  • the first pair of disulfide bonds were first formed between the two cysteines 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 the cysteic acid, at the same time A second pair of disulfide bonds are formed between the cysteines.
  • the bicyclic peptide was purified by reverse-phase HPLC C18 column (Vydac) to obtain ocO-conotoxin which was disulfide-bonded between the corresponding cysteines in order from N-terminus to C-terminus, and passed through mass spectrometry ( MS) was identified as correct.
  • the theoretical molecular weight of the three isomers after oxidative folding was 3451.96 Da
  • the molecular weight of GeXIVA12 was 3451.83 Da
  • the molecular weight of GeXIVA13 was 3451.72 Da
  • the molecular weight of GeXIVA14 was 3452.05 Da.
  • Example 4 Expression of various subtypes of iiAChRs in rat, mouse and human in Ouchi Pond oocytes
  • rat neuronal nAChRs preparation of various subtypes ⁇ 3 ⁇ 2, 6 / 3 ⁇ 2 ⁇ 3, 6/ 3 ⁇ 4, 9 10, 4 ⁇ 2, 4 ⁇ 4, 3 ⁇ 4, 2 ⁇ 2, 2 ⁇ 4, ⁇ 7), human neuron nAChRs subtype ( 9 ⁇ ⁇ , 6/ 3 ⁇ 2 ⁇ 3, ⁇ 7 ), rat ⁇ Various mutants of 9 ⁇ 10 nAChR, and cRNA of mouse and human muscle type nAChRs (( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ), the concentration of which was measured by OD value at UV 260 nm.
  • a frog egg injected with cRNA was placed in a 30 uL Sylgard recording tank (4 mm X depth 2 mm) and gravity-infused with ND96 perfusate containing 0.1 mg/ml BSA (bovine serum albumin) (96.0 mM NaCl, 2.0 mM KC1) , 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 5 mM HEPES, pH 7.1-7.5) or ND96 (ND96A) containing 1 mM atropine at a flow rate of 1 ml/min. All conotoxin solutions also contained 0.1 mg/ml BSA to reduce non-specific adsorption of toxins.
  • BSA bovine serum albumin
  • Switching valves SmartValve, Cavro Scientific Instruments, Sunnyvale, CA
  • Huaxin toxin or Acetylcholine ACh
  • Free switching and a series of three-way solenoid valves 157 valves, Neptune Research, Northboro, MA
  • 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 (2000) position.
  • the glass electrode was drawn with a 1 mm outer diameter 0.75 inner diameter glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, FL) and filled with 3 M KC1 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 ⁇ M, 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.
  • IC 5 Q and error range are: GeXIVA12, 4.6 nM (3.18-6.65 nM); GeXIVA13, 22.7 nM (11.8-43.5 nM); GeXIVA14, 7 nM (3.6-13.4 nM).
  • the slope and error range of the isomer dose response curve are: GeXIVA12, 0.56 (0.44-0.69); GeXIVA13, 0.78 (0.29-1.26), GeXIVA14, 0.79 (0.23-1.36).
  • a 0-GeXIVA12 The nAChRs subtypes have varying degrees of blocking activity with a half-blocking dose of IC 5 . The slope of the dose response curve is shown in Table 2.
  • Table 2 O GeXIVA12 slope of the various subtypes of nAChRs half-block IC 50 dose and dose response curves
  • a O GeXIVA12 has a large difference in the blocking activity of ⁇ * ⁇ 4 and ⁇ * ⁇ 2 nAChRs, and the blocking activity of (X * ⁇ 2 nAChRs is much higher than that of ⁇ * ⁇ 4 nAChRs (Fig. 3, A - E); Mouse muscle type nAChR ( ⁇ ⁇ ) is closer to the blocking activity of oc 7 nAChR subtype.
  • ocO-GeXIVA12 prefers to block ⁇ A-containing nAChRs, including 6/ 3 ⁇ 2 ⁇ 3, 4 ⁇ 2, ⁇ 3 ⁇ 2 and ⁇ 2 ⁇ 2.
  • oc O GeXIVA12 The blocking activity of 9 10 nAChR is at least 56-663 times stronger than other subtypes. At a low concentration of 200 nM, oc O GeXIVA12 is a specific blocker of oc 9 oc 10 nAChR, and the blocking activity against other nAChRs subtypes is weak, or almost none.
  • the bioactivity of the target of oc O-GeXIVA12 with other superfamily conotoxins is shown in Table 3.
  • Table 3 Comparison of the properties of ⁇ -GeXIVA with other related conotoxins Toxins ft g called source species Sequence Point Molecular weight a9al0 nAChR »
  • oc 9 (x l0 nAChR has high permeability to calcium ions (Ca ++ ).
  • Calcium ion influx can be activated by calcium influx of nAChRs (CD outflow current, on Xenopus oocytes, This current accounts for more than 90% of the observed oc 9 (x l0nAChR open current.
  • cesium ion (Ba ++ ) close to the calcium ion does not activate the chloride ion current.
  • IC 50 and the error range is 37 nM (25.0-55.7 nM), the slope of the curve ( Hillslope ) and the error range is 0.54 (0.42-0.65); isomers with I-IV; II-III disulfide bond a O GeXIVA14 has a half-blocking dose (IC 5 ) for oc 9 oc 10 nAChR and an error range of 5.8 nM (4.7-7.1 nM) with a slope of the slope and an error range of 0.65 (0.56-0.73).
  • Xenopus laveis oocytes were dissected and injected into the frog eggs. Each subunit was injected in 5 ng cRNA. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection, and voltage clamp recording of NMDA receptor was performed within 1-5 days after injection. The voltage of the NMDA receptor was recorded by voltage clamp using the same recording method as nAChRs, except that the perfusion solution used was a magnesium-free ND96 perfusion solution (Mg 2+ -free ND96 buffer) with a composition of 96.0 mM NaCl, 2.0 mM.
  • Mg 2+ -free ND96 buffer magnesium-free ND96 perfusion solution with a composition of 96.0 mM NaCl, 2.0 mM.
  • the NMDA receptor agonist solution is Mg 2+ -free ND96cho a O GeXIVA12 with a final concentration of 200 ⁇ M glutamic acid ( glutamate 20 ⁇ M glycine), which has the strongest blocking activity against the NR2C NMDAR subtype. The elution rate is faster.
  • O GeXIVA12 has a half-blocking dose (IC 5 ) for four NMDA receptor subtypes and the error ranges are: NR2C, 0.66 ⁇ ⁇ (0.38-1.1 ⁇ M); NR2B, 4.0 ⁇ ⁇ ( 2.2-7.3 ⁇ ⁇ ); NR2A, 3.7 ⁇ ⁇ (2.8-5.0 ⁇ M); NR2D, 5.2 ⁇ ⁇ (1.7-15.7 ⁇ M); O GeXIVA12 dose response curve slope of four NMDA receptor subtypes ( Hillslope ) And the error ranges are: NR2C, 0.13 (0.42-0.97); NR2B, 0.22 (0.36-1.3); NR2A 0.10 (0.61-1.07); NR2D, 0.15 (0.10-0.78) (Fig. 5).
  • a O-GeXIVA III The half-blocking dose of the isoforms for various MDARs subtypes IC 5 and the slope of the dose response curve are listed in Table 4.
  • O GeXIVA12 against the four NMDA receptor subtypes from strong to weak is: NR2C > NR2A > NR2B > NR2D.
  • Oc O GeXIVA14 has weak blocking activity against two NMDA receptor subtypes NR2B and NR2A, while oc 0-GeXIVA13 has weak or no blocking activity against four NMDA receptor subtypes.
  • Example 7 o O-conotoxin GeXIVA Three isomers block other rat nAChRs and mouse muscle type nAChRs
  • the blocking activity of GeXIVA14 is the strongest, and the IC 5 for a 2 p 2 is only 122 nM, The IC 5 of ⁇ 4 ⁇ 2 is 200 nM.
  • the blocking activity of ⁇ 3 ⁇ 4, a 6/ a 3 ⁇ 4 nAChR the blocking activity of GeXIVA13 is the strongest, and its IC 5 is 483 nM; the blocking activity of GeXIVA14 The IC 5 is 611 nM; GeXIVA12 has the weakest blocking activity, and its IC 5 is 806 nM.
  • the three isomers have similar blocking activity on human muscle type nAChR (Human ⁇ ⁇ ), and their half-blocking doses 1 ⁇ 5() are: a O GeXIVA12, 497 nM; O-GeXIVA13, 485 nM; O-GeXIVA14, 365 nM. However, for blocking activity with the a7 nAChR subtype, the difference between the three isomers is 4 ⁇ , and its IC 5 .
  • the sequence of activity is a O GeXIVA12, 555 nM > a O GeXIVA14, 865 nM > a O GeXIVA13, 3300 nM.
  • Blocking activity sequence for human a 9 a 10 and a 7 nAChRs O GeXIVA12 > GeXIVA14 > GeXIVA13 with rat a 9 a 10 is consistent with the order of blocking activity of a 7 nAChRs.
  • the blocking activity of the three isomers on human 6/3 ⁇ 2 ⁇ 3 nAChR is a O GeXIVA13 ⁇ GeXIVA14 > GeXIVA12, their half-blocking dose IC 5 . They are: O GeXIVA13, 141 nM; O-GeXIVA14, 197 nM; O-GeXIVA12, 505 nM.
  • a variety of mutants of the a 9 a 10 nAChR receptor were prepared by point mutation of a possible key amino acid at the binding site of conotoxin to the wild type a 9 a 10 nAChR receptor, ie, a single amino acid change, mutation
  • the site is indicated in Figure 8, for example, "roc9R71G (xlO,) represents the arginine R (Arg) mutation at position 71 on the a 9 subunit of the rat (r) wild type a 9 a 10 nAChR receptor.
  • G glycine acid
  • the mutant mutant is produced by PCR, mutating the a 9 or a 10 subunit gene of the wild type a 9 a 10 nAChR receptor. Corresponding to the codons encoding the point mutation amino acid.
  • the obtained mutants were studied in the same experimental manner as in Examples 4 and 5, and the three isomers of ocO-conotoxin GeXIVA were blocked to block various a.
  • the role of the 9 a 10 nAChRs mutant ( Figure 8).
  • the blocking activity of the three isoforms a O GeXIVA12, a O GeXIVA13 and a 0-GeXIVA14 on 11 rat oc9 (x10 nAChR receptor mutants was tested.
  • the concentration dose response curve is shown in Figure 8 (AK).
  • the general trend is that three isomer pairs 9 10 nAChR mutants have a strong blocking activity, O GeXIVA12 and oc 0-GeXIVA14 have similar blocking activities for various mutants, and are more active than ⁇ O GeXIVA13, which is related to wild type
  • the blocking activity of ⁇ 9 ⁇ 10 nAChR is consistent with the order.
  • the blocking activity sequence of the three isomers was oc 0-GeXIVA12 ⁇ GeXIVA14 > GeXIVA13. There were 5 mutants with a large change in activity, and a half-blocking dose of a O GeXIVA12 IC 5 . Between 46-59 nM; a O GeXIVA14 half-blocking dose IC 5 . Between 34-96nM; O GeXIVA13 between the semi-blocking dose IC 50 106-232nM. 3 isomers for these 5 (X 9 (X 10 nAChR mutant semi-blocking dose IC 5 .
  • a O GeXIVA12 blocks the elution of a 9 a 10 nAChR (Fig. 9A).
  • a -CTX RglA [S4T; R9Cit; Y10Iodo, RllQ] (referred to as RglAM) is a specific blocker of a 9 a 10 nAChR, but the elution recovery is very slow (experimental), that is, when the toxin RglAM is eluted, 9 l0 nAChR returns to ACh Normal gated open state is very slow ( Figure 9B).
  • the inventors designed a competitive experiment based on the difference in elution speed between the two (Fig. 9C). That is, first blocking oc9 with 1 ⁇ ocO-GeXIVA12 (xl0 nAChR lmin, then continue incubation with 20 nM a-CTX RglAM and 1 ⁇ a 0-GeXIVA12 in the cell slot of the same frog egg to block a 9 a 10 nAChR 5 min.
  • the Ach gated current recording showed that the elution rate was very slow, and it was the same as that of 20 nM -CTX RglAM alone.
  • ND96 was set to replace a O GeXIVA12 and a-CTX RglAM, respectively, as a positive and negative control. It was shown that ocO-GeXIVA12 could not prevent the oc-CTXRgIAM pair (X9 (X 10 nAChR blocking effect), which proved that the binding sites of a 9 a 10 nAChR were completely different, and a 0-conotoxin GeXIVA12 bound a 9 a 10 nAChR novel sites, and The binding sites of the previously discovered oc-conotoxin were different and did not overlap.
  • oc 9 x l0 nAChR is a new point in the treatment of neuralgia, cancer chemotherapy, breast cancer, lung cancer, wound healing, etc.
  • Example 11 Recombinant a 0-conotoxin GeXIVAWT inhibits the growth of Sf9 cells
  • the wild type toxin ( oc O-GeXIVAWT ) gene was inserted into the cleavage site Nco I and Xho I of the E.
  • coli expression vector pET22b (+) to construct a N-terminal fusion pelB leader and a C-terminal His-tag purification.
  • Isolation, purification and preparation of oc O-GeXIVAWT recombinant protein were carried out in a conventional manner.
  • the effect of recombinant conotoxin oc O-GeXIVAWT on the growth of Sf9 cells (Sopenoptera frugiperda 9 (Sf9) cells, purchased from Invitrogen, USA) was studied by MTT assay (Fig. 10). The method is based on MTT, which can enter the cell through the cell membrane.
  • the amber dehydrogenase in the living cell mitochondria can reduce the exogenous MTT to the blue-purple needle-shaped formamidine crystal which is hardly soluble in water and deposit in the cell, and die. Cells do not have this function.
  • Dimethyl sulfoxide (DMSO) dissolves the blue-violet crystals deposited in the cells, and the color of the solution is proportional to the amount of formazan contained.
  • the light absorption value (OD value) is measured by a microplate reader at a wavelength of 570 nm, which indirectly reflects the number of cells.
  • the Sf-9 insect cell line is derived from the ovarian cell line Sf-21 of the agricultural pest Lepidoptera Frugiperda, which is highly susceptible to the ⁇ California nuclear polyhedrosis virus (AcMNPV baculovirus) as a biological pesticide. ) Infected and can be used with all baculovirus expression vectors.
  • the wild-type recombinant conotoxin oc O-GeXIVAWT has a good application potential in pest control (Bruce C, Fitches EC, Chougule N, Bell HA, Gatehouse JA (2011) Recombinant conotoxin, TxVIA, produced in yeast has insecticidal Activity. Toxicon 58:93-100 ⁇ ).

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Description

α θ-超家族芋螺毒素肽、 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 oc O-超家族芋 螺毒素肽、 其药物组合物、 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烟碱乙酰胆碱受体（nAChRs )或 NMDA受体（NMDAR)的方法、 以及所述芋螺毒素肽的制药用途。 背景技术
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， 其介导众多中枢和外周神经系统 的生理功能， 包括学习、 记忆、 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、 Y -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， 这些疾病包括成瘾、 疼痛、 癌症、 智障、 帕金森症、 精神病、 抑郁、 重症肌无力等疑难杂症。 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 nAChR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. Nicke, A., Wonnacott, S. & Lewis, R. J. Alpha-conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. European journal of biochemistry / FEBS 271， 2004， 2305-2319. Dani, J. A. & Bertrand, D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annual review of pharmacology and toxicology 2007,47:699-729 ) 。
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， 或直接作为相关疾病的治疗药物， 或作为 工具药来研究和鉴定各种亚型的精细组成和生理功能。 此外， 在乳腺 癌与肺癌等组织中， 肿瘤细胞膜上烟碱乙酰胆碱受体的激活促进肿瘤 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（X和 p亚基组装成多种亚型， 每种亚型都有截 然不同的药理学特征。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。
研究表明，（X 9 (X 10 nAChR是治疗神经痛药物的新靶点 (Mcintosh. J. Μ·; Absalom, N.; Chebib, Μ·; Elgoyhen, A. B.; Vincler, M.， Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochemical pharmacology 2009， 78 (7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z.， Alpha-conotoxin Vcl.l alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain research 2005, 1059 (2), 149-58·)。 oc 9 oc 10 nAChR阻断剂具有治疗神经痛和加速受伤神经恢复的功能，可能是通 过免疫机制发挥作用（ Holtman, J. R.; Dwoskin, L. P.; Dowell, C; Wala, E. P.; Zhang, Ζ·; Crooks, P. A.; Mcintosh, J. M.， The novel small molecule alpha9alphal0 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic. European journal of pharmacology 2011， 670 (2-3), 500-8. Zheng, G.; Zhang, Ζ·; Dowell, C; Wala, E.; Dwoskin, L. P.; Holtman, J. R.; Mcintosh, J. Μ·; Crooks, P. A., Discovery of non-peptide, small molecule antagonists of alpha9alphal0 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain. Bioorganic & medicinal chemistry letters 2011， 21 (8)， 2476-9. )角化细胞上的 oc 9 oc 10 nAChR在 伤口愈合的病理生理学过程中起着很重要的作用 （ Chernyavsky, A. I.; Arredondo, J.; Vetter, D. E.; Grando, S. A., Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Experimental cell research 2007, 313 (16), 3542-55 ) 。 新近研究表明， a 9 nAChR亚基在乳腺癌 组织中过表达。 oc 9亚基变体影响支气管细胞的转化与增殖， 该亚基 在肺癌的治疗中具有非常重要的意义（Chikova, A.; Grando, S. A.， Naturally occurring variants of human Alpha9 nicotinic receptor differentially affect bronchial cell proliferation and transformation. PloS one 2011, 6 (11), e27978. ) 。
据调查， 疼痛影响 1/6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。 现有治疗神经痛的方法， 主要是局部麻 醉用药， 来阻断由于外周神经、 神经丛、 背根神经、 交感神经系统等 产生的疼痛信号。 但这些治疗只能短时间有镇痛效果， 但并不能根治 神经痛。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、 骨髄瘤、 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。
以 a 9 a 10 nAChR为靶点的神经痛治疗药物可通过肌肉注射发挥 镇痛效应（Vincler, M. Wittenauer, S. Parker, R. Ellison, M. Olivera, B. M. Mcintosh, J. M. Molecular mechanism for analgesia involving specific antagonism of alpha9alphal0 nicotinic acetylcholine receptors. Proc Natl Acad Sci U S A, 2006,103 ( 47 ) : 17880-4·)， 比目前商业化的 ω -CTX MVIIA镇痛药物-奇考诺肽给药途径更简便， 奇考诺肽需通过编程泵内 置于人体内直接给药至脊髄， 给药途径很麻烦， 该给药泵非常昂贵， 目 前仅限于美欧等发达国家应用， 很难在广大的发展中国家使用 （Kress HG, Simpson KH， Marchettini P， Ver Donck A, Varrassi G. Intrathecal therapy: what has changed with the introduction of ziconotide. Pain Pract. 2009; 9(5) :338-47. Burton AW, Deer TR， Wallace MS, Rauck RL, Grigsby E. Considerations and methodology for trialing ziconotide. Pain Physician. 2010;13(l):23-33. Wallace MS, Rauck RL, Deer T. Ziconotide combination intrathecal therapy: rationale and evidence. Clin J Pain. 2010;26(7):635-44 ) 。
Figure imgf000005_0001
(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. ) 。 多种 nAChRs亚型不但是尼 古丁成瘾的药物作用靶点， 还是滥用药物吗啡、 可卡因等成瘾的药物 作用靶点。
NMDA受体 ( N-methyl-D-aspartate receptor )是中枢神经系统内 一类重要的兴奋性 ^酸受体， 是一类配体门控离子通道型受体， 具 有广泛的生理学和药理学意义。 NMDA受体不仅在神经系统发育过程 中发挥着重要的生理作用， 如可调节神经元的存活， 调节神经元树突、 轴突结构发育及参与突触可塑性的形成等； 在神经元回路的形成中 NMDA受体亦起着关键作用。 在局部缺血集中部位， NMDA受体被激 活，使细胞外钙离子内流，从而导致细胞死亡（Twede, V. D.，Miljanich， G.， Olivera, B. M. & Bulaj, G. Neuroprotective and cardioprotective conopeptides: an emerging class of drug leads. Current opinion in drug discovery & development 2009， 12: 231-239)。 通过大鼠实验的研究表 明， NMDA 受体主要分布在中枢系统中， 如大脑、 脊髄； NMDA受体 在外周也有分布， 如 NR3B主要在运动神经元处表达， 而外周 NMDA 受体在面部肌肉痛感以及水肿形成中起到了很重要的作用。
研究表明， NMDA受体是学习、 记忆、 疼痛等过程中一类至关重 要的受体， 也是诸多神经疾病发病与治疗的作用靶点， 包括顽固性疼 痛、 药物和酒精成瘾、 瘫痫症、 局部缺血、 帕金森症、 痴呆、 兴奋性 神经元细胞死亡等等 （Sattler, R. et al. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science (New York, N. Y.) 1999， 284, 1845-1848. Lewis, R. J. & Garcia,
M. L. Therapeutic potential of venom peptides. Nature reviews. Drug discovery, 2003, 2: 790-802. Sheng, Z.， Liang, Z.， Geiger, J. H.，
Prorok, M. & Castellino, F. J. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B. Neuropharmacology , 2009， 57, 127-136. Meldrum, B. S. The role of glutamate in epilepsy and other CNS disorders. Neurology, 1994,44: SI 4-23. Ulas, J. et al. Selective increase of NMDA-sensitive glutamate binding in the striatum of Parkinson's disease, Alzheimer's disease, and mixed Parkinson's disease/ Alzheimer ' s disease patients: an autoradiographic study. The Journal of neuroscience, 1994， 14， 6317-6324. Ozawa, S.， Kamiya, H. & Tsuzuki, K. Glutamate receptors in the mammalian central nervous system. Progress in neurobiology, 1998,54: 581-618. Bisaga, A. & Popik, P. In search of a new pharmacological treatment for drug and alcohol addiction: N-methyl-D-aspartate (NMDA) antagonists. Drug and alcohol dependence, 2000， 59: 1-15).
目前， 生活在热带海洋中的肉食性软体动物芋螺毒液中产生的毒 素 （芋螺毒素， conopeptide, conotoxin, CTX )备受关注， 已被用于 系统地研究与开发 nAChRs各种亚型的特异阻断剂。
芋螺毒素 （conopeptide, conotoxin, CTX ) 大多是由 7 - 50个氨 基酸残基组成的、 富含半胱氨酸（Cys ) 的神经肽毒素。 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 18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 ( Kaas Q, Yu R， Jin AH, Dutertre S, Craik DJ (2012) ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res 40: D325-330. 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 a 9 a 10 Nicotinic Acetylcholine Receptors from Conus vexillum Delineates a New Conotoxin Superfamily. PLoS ONE, 8(1): e54648 (1-10)， 2013 ) 。 芋螺毒素 （肽）按其受体靶位可分为 α、 ω、 μ、 δ等多种药理学家族。 其中的 a类芋螺毒素 （ oc *-CTX )具有阻 断烟碱乙酰胆碱受体 (nAChRs)的功能； 不含有半胱氛酸的芋螺毒素肽 Conantokins 具有阻断 N-甲基 -D-天冬氨酸受体 （ NMDA 受体， N-methyl-D-aspartic acid receptor, NMDAR)的特殊功能。 每个超家 族芋螺毒素根据受体靶类型， 又可分为 oc、 oc A、 Κ Α ( Α-超家族） ， ω、 δ、 κ、 μ Ο ( 0-超家族） ， μ、 φ、 κ Μ ( Μ-超家族）等家族
(亚型） 。
芋螺毒素具有特异结合动物体内各种离子通道的特殊功能。 目前， 芋螺毒素备受关注， 已被用于系统地研究与开发 nAChRs各种亚型的 特异阻断剂。 发明内容
本发明人经过深入的研究和创造性的劳动， 发现了一类新的 oc O- 超家族芋螺毒素肽。 本发明人惊奇地发现， 本发明的 oc O-超家族芋螺 毒素肽能够特异性地阻断乙酰胆碱受体和 NMDA受体，特别是对神经 痛药物靶点、 乳腺癌、 肺癌靶点 oc 9 (x l0 nAChR的阻断活性最强， 具 有在制备镇痛药物， 抗成瘾、 瘫痫、 或癌症的药物， 以及神经科学工 具药等方面的良好应用前景。 由此提供了下述发明： 本发明的一个方面涉及一种多肽， 其为或者包含一个或多个相同 或者不同的选自如下的 （1 )至 （3 ) 中任一项所述的氛基酸序列： ( 1) SEQ ID NO: 7- 12中任一序列所示的氛基酸序列；
( 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 )所述序列有所 不同的赛基酸序列。
其中， SEQ ID NO: 7或 8是命名为 GeXIVAWT的野生型的前体 肽； SEQIDNO: 9是其成熟肽。
SEQ ID NO: 10或 11是命名为 GeXIVA的突变型的前体肽； SEQ ID NO: 12是其成熟肽。
GeXIVA及其野生型 GeXIVAWT的前体肽含有信号肽、 前肽和成 熟肽 3个区域， 它们的具体序列及其分析如实施例 1-2中所述。 野生 型成熟肽具有 5个半胱氨酸，与以前发现的所有芋螺毒素的偶数个半胱 氨酸模式都不同， 是一个 ocO-超家族新芋螺毒素， 由于该毒素阻断 oc9 lO nAChR 的活性最强， 所以命名为 ocO-超家族芋螺毒素 （ a O-conotoxin ) 。
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 [查询文档卜 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 列与 SEQ ID NO: 1-4中任一 J ^^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 /或活性的保守性氛基酸取代； 小片段缺失， 通常是 1到大约 5个、 优选 1-3个、 更优选 1个氨基酸的缺失； 小的氛基或羧基末端 延伸， 如氨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 多聚组氨酸片段、 抗原表位或结合区。
不拘于理论的限制， 保守性取代的例子是在碱性氛基酸 (精氨酸、 赖氨酸和组氨酸)、 酸性氛基酸 (谷氨酸和天冬氨酸)、极性氨基酸 (谷氨 酰胺和天冬酰胺)、 疏水氛基酸 (亮氨酸、 异亮氨酸和纈氨酸)、 形状相 似的氛基酸（精氨酸和丝氨酸之间） 、 与半胱氨酸密码子相差一个碱 基的氛基酸（半胱氨酸、 精氨酸、 丝氨酸、 色氨酸、 甘氨酸、 酪氨酸 酸 (甘氨酸、 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氨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, Arg/Ser, Cys/Ser, Arg/Cys, Arg/Ala, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu和 Asp/Gly等以及反向进行的替换。
本发明还包括在本发明 oc O-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， 其中， SEQ ID NO: 12中的 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； 或者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； 或者第一个半胱氨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； 具体地， 所述多肽的羧基末 端是自由的 C-末端， 或者是酰胺化的。 本发明的上述多肽即为芋螺毒素肽；具体地，是 (Χ θ-芋螺毒素肽。 上述芋螺毒素肽可以从我国海南产的将军芋螺 ( ins C. generalis) 中提取， 或将其基因进行推导获得成熟肽序列。 也可以化学合成氛基 酸序列 （例如参考实施例 3中的方法） ； 或者通过基因重组的手段表 达其核苷酸（核苷酸序列的制备参考实施例 1-2或者直接人工合成 )， 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(Cys), But (Thr、 Ser、 Tyr),OBut (Asp),Boc (Lys); 半胱氨酸用 Trt或 Acm保护基团， 分别在 相应的半胱氨酸之间定点形成二硫键；或所有半胱氨酸都用 Trt保护基 团， 通过一步法氧化折叠随机形成二硫键。
2)将步骤 1 ) 中得到的线性多肽从树脂上切割下来， 并用冰乙醚 沉淀和洗涤回收线性多肽粗品， 用制备型反向 HPLC C18 柱（Vydac) 纯化；
3)将步骤 2) 中得到的产物进行两步或一步氧化折叠。 本发明的另一方面涉及一种多核苷酸， 其编码本发明任一项所述 多肽的赛基酸序列。
根据本发明任一项所述的多核苷酸， 其为包含或者一个或多个相 同或不同的选自如下的 （1 )至 （3) 中任一项所述的核苷酸序列：
( 1 ) SEQ ID NO: 1 - 6中任一序列所示的核苷酸序列；
( 2 ) SEQ ID NO: 1 - 6中任一序列的互补序列； 或
(3)在严谊条件下能够与上述（1 )或（2)中所述的核苷酸序列 杂交的核苷酸序列。
SEQ ID NO: 1 - 6分别是 SEQ ID NO: 7- 12的编码序列。
其中， SEQ ID NO: 1或 2编码命名为 GeXIVAWT的野生型的前 体肽； SEQ IDNO: 3编码其成熟肽。 SEQ ID NO: 4或 5编码命名为 GeXIVA的突变型的前体肽； SEQ ID NO: 6编码其成熟肽。
SEQ ID NO: 1 - 6具体序列及其分析如实施例 1 - 2中所述。
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*Ό下在 6Χ SSPE、 5Χ Denhardt氏溶液、 0.1 % SDS、 O.lmg/ml变性 DNA中进行过夜。 清洗 通常如下进行：于 Tm O 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引物对， 其为或者包含 SEQ ID NO: 13和 SEQ ID NO: 14所示的核苷酸序列。 所述引物对能够用于 PCR 扩增， 得到 SEQ ID NO: 1或 SEQ ID NO: 2所示的多核苷酸序列。 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重组表达载体， 其包含本发明的核酸 构建体； 具体地， 所述重组载体是重组 pET22b(+)载体。
本发明的再一方面涉及一种转化的细胞， 其包含本发明的重组表 达载体； 具体地， 所述转化的细胞为转化的 Sf9细胞。
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组合物， 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和 NMDA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者融合蛋白的步 骤； 具体地， 所述乙酰胆碱受体是 (X 9 (X 10乙酰胆碱受体， 或其他乙酰 胆碱受体亚型和 NMDA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和 NMDA 受体或者确定乙酰胆碱受体和 NMDA受体亚型的方法， 该方法包括： 通过在存在和不存在候选化合物存在的情况下将乙酰胆碱受体或 NMDA受体亚型与本发明任一项所述的多肽或者融合蛋白进行接触 的步骤； 具体地， 所述乙酰胆碱受体为（X 9 (X 10乙酰胆碱受体； 或所述 NMDA受体为 NR2C NMDA受体。 当 oc 0-芋螺毒素 GeXIVA在很低浓 度下能够特异阻断 a 9 a 10乙酰胆碱受体时，则推断该乙酰胆碱受体是 a 9 a 10亚型的乙酰胆碱受体； 或当 oc O-芋螺毒素 GeXIVA 在较低浓 度下能够特异阻断 NR2C NMDA受体时， 则推断该 NMDA受体是 NR2C匪 DA受体。 本发明的再一方面涉及本发明任一项所述的多肽或者融合蛋白用 于阻断乙酰胆碱受体和 NMDA受体的用途； 具体地， 所述乙酰胆碱受 体是 (X 9 (X 10乙酰胆碱受体， 或其他乙酰胆碱受体亚型； 以及 NR2C NMDA受体或其他亚型。
本发明的再一方面涉及本发明任一项所述的多肽或者融合蛋白在 制备阻断乙酰胆碱受体和 NMDA受体的药物或试剂中的用途； 具体 地，所述乙酰胆碱受体是 a 9 10乙酰胆碱受体，与 NR2C NMDA受体。
本发明的再一方面涉及本发明任一项所述的多肽或者融合蛋白在 制备治疗或预防神经系统疾病例如神经痛、 乳腺癌、 肺癌、 成瘾、 瘫 痫症、 局部缺血、 帕金森症、 痴呆、 或兴奋性神经元细胞死亡等的药 物， 或者用于制备杀灭害虫 （例如鳞翅目秋粘夜蛾） 、 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、 骨髄瘤、 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 统疾病例如神经痛、 乳腺癌、 肺癌、 成瘾、 瘫痫症、 局部缺血、 帕金 森症、 痴呆、 或兴奋性神经元细胞死亡等的方法， 或者一种杀灭害虫 (例如鳞翅目秋粘夜蛾） 、 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）或者融合蛋白或者本发 明的药物组合物的步骤； 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、 骨髄瘤、 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 (X 9 (X 10 乙酰胆碱受体 (nAChR) 或 /和 NR2C NMDA受体发挥作用， 具有镇痛活性。 可应用于研究、 诊断和治疗神经痛、 乳腺癌、 肺癌、 帕金森症、 痴呆、 成瘾、 瘫痫症、 局部缺血等神经系统疾病、以及作为有用的分子探针用于研究等方面。 不同的（X类 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 (X类 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 nAchR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、 骨髄瘤、 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。
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个 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组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 ^ ^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和重组核酸序列。 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。 应用重组 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 的所有组分。 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 以 供带接头的调控序^。 术语 "可操作连接" 在文中定义为这样二 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 于本发明。
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 控序列的其他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 号的重组表达载体。 可以将上述各种核酸和调控序列连接在一起来制 备重组表达载体， 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 ^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。
重组表达载体可以是任何便于进行重组 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， 将整合到基因组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 基因组的全部 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组 中， 或保证载体在细胞中独立于细胞基因组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^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组中， 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 ^^列的细胞。
用于连接上述各元件来构建本发明所述重组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， 紐约， 1989)。
宿主细^
本发明还涉及包含可用来重组生产多肽的本发明所述核酸序列的 重组宿主细胞。 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
本发明还涉及重组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组成 (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)。
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组微生物应 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 组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株处理应用。 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 /mg。 这些制剂 将以每公顷大约 50mg (液体的或干的）一 lkg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。
药物组合物
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 物组合物。 所述药物组合物可用于研究、诊断、緩解或治疗与神经痛、 乳腺癌、 肺癌、 智障、 成瘾、 疼痛、 帕金森症、 精神病、 抑郁、 重症 肌无力、 瘫痫症、局部缺血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组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、 给药曰 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组合物非肠道给药、 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、 胶囊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或 /和 NMDA受体的药物组合物。 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或 /和 NMDARs的种系发生； 作为分子探针来确定 nAChRs 或 /和 NMDARs的不同亚型；作为分子模型，设计新药;作为研究、诊断 神经性疾病如帕金森氏病、 行动障碍、 精神分裂症、 瘫痫症、 局部缺 血等的工具药和治疗药物； 治疗神经痛、 成瘾、 乳腺癌、 肺癌、 小细 胞肺癌、 瘫痫症、 局部缺血等的侯选药物。 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 a 0-芋螺毒素肽能够特异地阻断乙酰胆碱受体 ( nAChRs )和 NMDA受体， 并且具镇痛活性和抑制乳腺癌和肺癌细 胞生长的功效， 以及治疗成瘾、 瘫痫症与局部缺血等的功效。 附图说明
注： 若图中没有标明的各种 nAChRs与 NMDA受体的亚型来源， 均为大鼠的相应受体， 图注和图中对大鼠的受体类型来源进行省略。
图 1: 显示的是 oc O-GeXIVA成熟肽序列 （SEQ ID NO: 1 ) 以及 具有 3种可能的二硫键连接方式的异构体。其中 GeXIVA12的二硫键连 接方式是 1-11， III-IV; GeXIVA13 的二疏键连接方式是 1-111， II-IV; GeXIVA14的二硫键连接方式是 I-IV, 11-111。
图 2: A显示的是 33 nM a O GeXIVA12对 oc 9 oc 10 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 33 nM α O GeXIVA12温育 5分钟后，第一个 Ach脉冲形成的电流轨迹（ ~ 0 nA ). Ach脉冲时间为 Is; 纵坐标是电流大小， 单位为 nA， 横坐标为累计 时间，单位为 ms，温育前后每相邻 2个电流轨迹之间相隔时间为 60s。 33 nM a O GeXIVA12完全阻断了 oc 9 oc 10 nAChR电流， 且洗脱速度 很快。图中 B、 C、 D分别为 3种异构体 a 0-GeXIVA12、 a 0-GeXIVA13、 ocO-GeXIVA14对 (x9(xl0nAChR的浓度剂量反应曲线。 图 B、 C、 D 中横坐标为所用 ocO-GeXIVA 异构体的摩尔浓度 （M) 的对数值 ( Log [Toxin Concentration] 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，每个剂量反应百分数为 6-12个非洲爪蟾卵母细胞记录 的数据平均值（mean) ， 曲线同时显示标准误（ SEM ) 。
图 3: oc O GeXIVA12对其他 nAChRs 亚型的浓度剂量反应曲线 (A-E)。 图中横坐标为所用 ocO-GeXIVA12 的摩尔浓度（M) 的对数 值 （Log[Toxin Concentration] 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，每个剂量反应百分数为 6-12个非洲爪塘卵母细胞记录 的数据平均值（mean)， 曲线同时显示标准误（ SEM )。 图上标示了 相应的 nAChRs 亚型以及对该亚型的半阻断剂量 （ IC5。） 。 a O GeXIVA12对 oc*p4 与 oc*p2 nAChRs的阻断活性差异较大， 对 a * β 2 nAChRs 的阻断活性远高于对 oc * β 4 nAChRs; 对小鼠肌肉型 nAChR (Μαΐβΐδ ε )与 oc7 nAChR亚型的阻断活性较接近。
图 4: 显示的是用钡离子 ND96灌流液（Ba++ -ND96 )代替常规 ND96灌流液时， 3种异构体 ocO-GeXIVA12， GeXIVA13与 GeXIVA14 对 OC90C10 nAChR 的浓度剂量反应曲线， 图中横坐标为所用 3 个 oc O-GeXIVA 异构体的摩尔浓度 （ M ) 的对数值 （ Log[Toxin Concentration] 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。 每 个剂量反应百分数为 9 个非洲爪蟾卵母细胞记录的数据平均值 (mean) , 曲线同时显示标准误（ SEM )。 图上标示了相应的异构体 对 oc 9 oc 10 nAChR 亚型的半阻断剂量（IC5。） 。
图 5: oc O GeXIVA12对 NMDA受体各种亚型的浓度剂量反应曲 线。 图中横坐标为所用 oc O-GeXIVA12 的摩尔浓度（M ) 的对数值； 纵坐标为剂量反应百分数（％ Response ) ， 是相应浓度的毒素作用下 NMDA 受体电流与对照电流的比值百分数， 每个剂量反应百分数为 3-5个非洲爪塘卵母细胞记录的数据平均值（mean ) ， 曲线同时显示 95%置信区间的标准误 ( SEM ) 。 图上标示了相应的 NMDARs亚型 以及对该亚型的半阻断剂量（IC5。） 。
图 6:异构体 0-GeXIVA12、 O GeXIVA13与 O GeXIVA14 对 nAChR受体其他各种亚型的浓度剂量反应曲线 (A- J)。 图中各种标 示含义与图 3相同。
图 7 : 3 种异构体 a O GeXIVA12、 a O GeXIVA13 与 a O GeXIVA14 对人类 nAChR 受体有关亚型的浓度剂量反应曲线 (A-F)。 图中各种标示含义与图 3相同。
图 8 : 3 种异构体 a O GeXIVA12、 a O GeXIVA13 与 a 0-GeXIVA14对大鼠 oc 9 (X 10 nAChR受体的各种突变型的浓度剂量反 应曲线 (A-K)。 图中各种标示含义与图 3相同。
图 9: 显示的是 oc O-GeXIVA12阻断（x 9 (x l0 nAChR后， 不能阻 止 oc -CTX RgIA [S4T; R9Cit; YlOIodo, R11Q】对 oc 9 oc 10 nAChR的 阻断作用， 证明两者之间结合 oc 9 oc 10 nAChR的位点完全不同。 A. 1 μ Μ a O GeXIVA12 5 min; B. 20 nM a -CTX RgIA [S4T; R9Cit; YlOIodo, R11Q] 5 min; C. 1 μ Μ a 0-GeXIVA12 lmin + 【1 μ M a O-GeXIVA12+20 nM a -CTX RgIA [S4T; R9Cit; YlOIodo, R11Q】 5 min】 。
图 10: 显示的是重组 a O-超家族芋螺毒素野生型 GeXIVAWT ( rCTX-K41 )对 Sf9细胞的抑制效果。图中横坐标为重组 GeXIVAWT 的浓度，单位为 g/ml， Control为不加重组 GeXIVAWT的阴性对照； 纵坐标是在酶标免疫测定仪（ BIO-RAD MODEL 550) 上测定的 490 nm 波长下的吸光度值。 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: 新的 o O-超家族芋螺毒素基因野生型 （GeXIVAWT ) 的克隆和序列分析
以从海南岛、 西沙群岛等沿海采集的将军芋螺（ C. generalis )活 体为材料。用小量柱离心式组织 /细胞总 RNA抽提试剂盒 (上海华舜生 物工程有限公司）， 按操作手册提取总 RNA， 然后进行 cDNA的合成。 具体步骤可以参照文献（例如权娅茹， 罗素兰， 林秋金， 长孙东亭， 张本。 芋螺毒素 RNA的提取及其 cDNA合成的研究， 中国海洋药物， 2005， 24 ( 2 ) :1-5 )进行。
以上述合成的 cDNA为模版， 根据 01-基因超家族前体基因的非 翻译区序列设计引物， 进行 RT-PCR扩增， 获取特异性 PCR扩增产 物。 所用引物序列为：
引物 1: 5， -CATCGTCAAGATGAAACTGACGTG-3' (SEQ ID NO: 13);
引物 2: 5， -CACAGGTATGGATGACTCAGG-3' (SEQ ID NO:
14)。
RT-PCR循环程序为： 94 预变性 3分钟， 94 变性 30秒， 56 退火 30秒， 72 延伸 30秒， 30个循环后， 72 再延伸 2分钟。
回收上述特异 PCR产物，与 T-easy载体 (Promega)连接后转化大 肠杆菌 XL1菌株（也可以使用其它的商业化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组子， 抽提纯化重组子质粒用 于测序， 同一 PCR产物选取不同的克隆（例如 3 - 5个不同的克隆）进 行测序、 分析。
经序列分析比较， 获得了本发明所述的 01-超家族芋螺毒素新成 员， 即野生型前体肽 GeXIVAWT的 cDNA基因。 GeXIVAWT前体肽 基因经 DNAStar软件分析，获知其开放阅读框（ ORF )序列即 SEQ ID NO: 1和 SEQ ID NO: 2， 如下：
编码 GeXIVAWT前体蛋白的开放阅读框（ ORF ) (存在等位基因 变异， 加边框的第 19位 T与 G表示其单碱基突变位点； 下划线部分表 示编码成熟肽的 DNA序列） ：
ATGAAACTGACGTGCGTG@TGATCATCACCGTGCTGTTCC TGACGGCCTGTCAACTCACTACAGCTGTGACTTACTCCAGAGG TGAGCATAAGCATCGTGCTCTGATGTCAACTGGCACAAACTAC AGGTTGCCCAAGACGTGCCGTAGTTCCGGTCGTTATTGTCGCT CACCTTATGATTGCCGCAGAAGATATTGCAGACGCATTACGGA
TG
Figure imgf000024_0001
TGACGGCCTGTCAACTCACTACAGCTGTGACTTACTCCAGAGG TGAGCATAAGCATCGTGCTCTGATGTCAACTGGCACAAACTAC AGGTTGCCCAAGACGTGCCGTAGTTCCGGTCGTTATTGTCGCT CACCTTATGATTGCCGCAGAAGATATTGCAGACGCATTACGGA TGCGTGCGTATAG ( SEQ ID NO: 2 )
上面的序列编码的 Ol-超家族芋螺毒素新成员， 即野生型前体肽 GeXIVAWT (下文中也称为 a O-conotoxin GeXIVAWT precursor或 Ο- GeXIVAWT precursor或 GeXIVAWT precursor )的氨基 ^^列 如下（加边框的第 7位 L与 V表示突变位点的氛基酸， 下划线部分表 示信号肽的氛基酸序列， 箭头 表示成熟肽前后翻译后修饰加工位点， 斜
Figure imgf000024_0002
LPK i TCRSSGRY CRSPYDCRRRYCRRITDACV ( SEQ ID NO: 7 )； 或 mKLTC\^YI\l^l,TACOl,TTAVTYSRGEHKHRALMSTGTNY RLPK i TCRSSGRY CRSPYDCRRRYCRRITDACV( SEQ ID NO: 8 )。
野生型 GeXIVAWT的前体肽 cDNA基因编码产生的信号肽和成熟 肽通 it ^线的 ProP 1.0 Server分 测 (Duckert, P.; Brunak, S.; Blom,
Ν·， Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection： PEDS 2004， 77 (1), 107-12·)。
编码 GeXIVAWT成熟肽的核苷酸序列如下 （加边框部分表示编 码半
Figure imgf000025_0001
TATAG ( SEQ ID NO: 3 ) 。
GeXIVAWT 野生型成熟肽 （下文中也称为 oc O-conotoxin GeXIVAWT 或 oc O- GeXIVAWT或 GeXIVAWT ) 的赛基酸序列如下 (加边框部分为半胱氨酸） ：
T@RSSGRY@RSPYD@RRRY@RRITDA@V ( SEQ ID NO: 9 ) „ 野生型 GeXIVAWT前体肽含有信号肽、 前肽和成熟肽 3个区域， 信号肽中第 7位的氛基酸残基为亮氨酸或纈氨酸（L或 V ) ， 对应的密 码子为 TTG或 GTG。 野生型成熟肽区域（SEQ ID NO: 9 )含有 5个 半胱氛酸（Cys ) ， 这与以往发现的芋螺毒素都不相同， 与相关基因超 家族的成员比较见表 1。
表 1: 与 αθ-基因超家族相关的芋螺毒素前体蛋白序列比较
称 半胱氨酸模 前体肽序列
式
aO-GeXI TVLFLTACQLTTA VTYSRGEHKHRAL VA wild c-c-c-c-c TCRSSGRYCRSPYDC RRYCRW type
M LTCVIL/VIHTVLFLTACQLTTAFTrSRG^H^H/ML aO-GeXI
C-C-C-C
VA 冒 歷 誦讀 TORSPYDR RRYCRRI
ITDACV
\1 LTCVV I VA V LLLTACQ LIT ADDSRGTQKHRALR
MVIIA C-C-CC-C-C STTKLSTSTRiC KG K (； Λ KC SR l.\IM)C C KiSC RS (； K
C|G
ME LTILLLVAAVLMSTQALI QSD GEKR Q QAKINFL
O TxVIIA C-C-CC-C-C ^RISIfAESWWEGECKGWSVYCSWDWECCSGEC
TRYYCELW
MSGLGIMVLTLLLLV FM EXSHQDAGEKQA TQRDAI
O CaFrl79 C -C -C C -C -C V VRRRRSLARRWW I： KC I： I： DC I： 1)1： KKH C CM N NG
PSCARLCFJG
PI PSVRSVTCCCLLVVM M FSVQLMPGSPGTA QLSG
J PIXIVA C-C-C-C R Ti R J I PR PR I C N I AC R A(i I (； H ΚΜ» I 11 C R I (； K R
DAVSSSMAV
M LSVMFIVFLM LT \1 PMTC A GISRSA TNGGEAD VR
L LtXIVA C-C-C-C 靈腦 M^lM^^i CKPSCTNCiG
<Ύ\ FLLW LJJJWSFPSDRASDGRNAAANDKASDl a-BuIA CC-C-C
//J AKiC C Sn 'C AV Ι J GRRR
A MFTXFLLWL AT JWSFTSDRASDDRNTNDKASRLL
aA-PIVA CC-C-C-C-C
S7/ 7M(;(((;SYl，\AA(ni，(S(KI)Kl，SY((;OI(;R
[VIQTAYWV.VIVMMMVMVVITAPLSEG7^P^X//R7L
C aC-PrXA C-C r V/ //W//, fSA/S6f Ι Ί (； l\ DAKI SC ACil.RC;
G —C—V—LP—P—N—LR—P—KF—K—E JGR
MPKLEMMLLVLLIFPLSYFIAAGGg WQ VDRRGDG aD-VxXX C-CC-C-CC
D A/>6)X0f67)f I DV ODC OV SH»(;SK\\ (； RC C I.NRV C
A C-C-C-C
GPMCCPASHCYCVYHRGRGHGCSC
M MS MGA \1 FVLLLLFT LASSQQEGD VQARKTHPK aS-RVIII C-C-C-C C- f^/ f
A C-C-C-C C 〃 fS(,7f | Κ( \ΊΊ)Κ( K (； T(;V、 \( (;I:S( S( I:
GLHSCRCTYNIGSMKSGCACICTYY
上表中， 字符底纹表示信号肽，斜体表示 N-末端前肽区， 下划线表 示成熟肽， 箭头丄表示成熟肽前后翻译后修饰加工位点； 加边框部分表 示突变位点氛基酸。 实施例 2: 新的 a 0-超家族芋螺毒素基因突变型 （GeXIVA) 的 制备和序列分析
野生型 GeXIVA WT前体肽基因的第 181 - 183 TGC编码半胱 氨酸 Cys， 对此进行点突变（也可通过直接人工化学合成 SEQ ID NO: 4获得） ， 即单碱基突变为编码精氨酸 Arg (R) 的密码子 CGC (@GC → GC)，即第 181位的 T变为 C (T181C)，该点突变体命名为 GeXIVA 前体肽基因， 其序列如下： 编码 GeXIVA前体蛋白的开放阅读框（ORF ) (存在等位基因变 异， 加边框的第 19位 T与 G表示其单碱基突变位点； 默下划线部分表 示第 181位的突变位点， 下划线部分表示编码成熟肽的 DNA序列 )：
ATGAAACTGACGTGCGTG@TGATCATCACCGTGCTGTTCC TGACGGCCTGTCAACTCACTACAGCTGTGACTTACTCCAGAGG TGAGCATAAGCATCGTGCTCTGATGTCAACTGGCACAAACTAC AGGTTGCCCAAGACGTGCCGTAGTTCCGGTCGTTATTGTCGCT CACCTTATGATCGCCGCAGAAGATATTGCAGACGCATTACGG AT
Figure imgf000027_0001
TGACGGCCTGTCAACTCACTACAGCTGTGACTTACTCCAGAGG TGAGCATAAGCATCGTGCTCTGATGTCAACTGGCACAAACTAC AGGTTGCCCAAGACGTGCCGTAGTTCCGGTCGTTATTGTCGCT CACCTTATGATCGCCGCAGAAGATATTGCAGACGCATTACGG ATGCGTGCGTATAG ( SEQ ID NO: 5 ) 。
上述序列编码 Ol-超家族芋螺毒素新成员， 即突变型前体肽 GeXIVA (下文中也称为 oc O-conotoxin GeXIVA precursor 或 oc O- GeXIVA precursor或 GeXIVA precursor ) 的 ^ ^^列 （加边框的 第 7位 L与 V表示突变位点的氛基酸，双下划线部分表示第 61位的人 工突变位点， 下划线部分表示信号肽的氛基酸序列， 箭头 表示成熟肽 前后翻译后修饰加工位点，斜体表示 N-末端前肽区 ) ；
LPK i TCRSSGRY CRSPYDRRRRYCRRITDACV( SEQ ID NO: 10 )。
MKLTC\\\UT \^\,TACQ\,TTAVTYSRGEHKHRALMSTGTNY
RLPK i TCRSSGRY CRSPYDRRRRYCRRITDACV ( SEQ ID NO: 11 ) 。
突变型 GeXIVA的前体肽 cDNA基因编码产生的信号肽和成熟肽 通过在线的 ProP 1.0 Server分 测（Duckert, P.; Brunak, S.; Blom, Ν·， Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection： PEDS 2004， 77 (1), 107-12·)。
编码 GeXIVA成熟肽的核苷酸序列如下 （加边框部分表示编码半 胱氨酸的密码子； 双下划线的字母 C表示点突变氛基酸对应的单碱基 突变
Figure imgf000028_0001
TATAG ( SEQ ID NO: 6 ) 。
GeXIVA突变型成熟肽（下文中也称为 a O-conotoxin GeXIVA 或 a O- GeXIVA或 GeXIVA ) 的氛基酸序列如下 （加边框部分为半胱氨 酸， 双下划线的字母 R表示点突变氛基酸） ：
T@RSSGRY@RSPYDRRRRY@RRITDA@V ( SEQ ID NO: 12 )。 突变型 GeXIVA前体肽含有信号肽、前肽和成熟肽 3个区域，信号 肽中第 7位的氛基酸残基为亮氨酸或纈氨酸（L或 V ) ， 对应的密码子 为 TTG或 GTG。 突变型成熟肽区域 ( SEQ ID NO: 12 )含有 4个半胱 氨酸（Cys ) ， 这与以往发现的芋螺毒素都不相同， 与相关基因超家族 的成员比较见上面的表 1。
下面的研究表明， GeXIVA是 nAChRs和 NMDARs的阻断剂， 且对 a 9 a 10 nAChR的阻断活性最强，所以正式命名为 oc O-conotoxin GeXIVA, 简写为 oc O- GeXIVA或 GeXIVA。 实施例 3: Ο-芋螺毒素 GeXIVA的人工合成
根据 Ο-芋螺毒素 GeXIVA成熟肽的氨基酸序列 （ SEQ ID NO: 12， C末端未酰胺化） ， 采用 Fmoc方法人工合成 GeXIVA三种可能 的异构体线性肽 GeXIVA12、 GeXIVA13、 GeXIVA14 (图 1 ) 。 具体 方法如下。
三个异构体的树脂肽采用 Fmoc化学方法进行人工合成， 除了半 胱氨酸外， 其余氨基酸用标准的侧链保护基团。 GeXIVA12的第 3和 第 4个半胱氛酸（ Cys ) 的 -SH用 Trt (S-trityl) 保护， 第 1和第 2个 半胱氨酸的 -SH用 Acm (S-acetamidomethyl) 成对保护； GeXIVA13 的第 2和第 4个半胱氛酸（ Cys ) 的 -SH用 Trt (S-trityl) 保护， 第 1 和第 3 个半胱氨酸的 -SH 用 Acm (S-acetamidomethyl) 成对保护； GeXIVA14的第 2和第 3个半胱氨酸（ Cys )的 -SH用 Trt (S-trityl) 保 护， 第 1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，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 ^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,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 10-50% B60. 溶剂 B是 60% ACN( acetonitrile )， 40 % H20， 0.92% TFA ( trifluoroacetic acid ) ； 溶剂 A是 1% 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， 其纯度达 96 %以上， 方可用于氧化折叠。 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 3个 异构体 GeXIVA12、GeXIVA13和 GeXIVA14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， 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 ocO-芋螺毒素， 并通过质谱 (MS)鉴定为正确。 氧化折叠后的 3个异构体的理论分子量 (monoisotopic mass)为 3451.96 Da, GeXIVA12的测定分子量为 3451.83 Da; GeXIVA13的测定分子量 为 3451.72 Da; GeXIVA14的测定分子量为 3452.05 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。 这些定量过的异构体继续用于后续的活性测试（例如实 施例 5-10 ) 。 实施例 4: 大鼠、 小鼠和人类 iiAChRs各种亚型在非洲爪塘卵母 细胞中的表达
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， 以及体夕卜转录试齐1 J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/ 3β2β3, 6/ 3β4, 9 10, 4β2, 4β4, 3 β 4, 2 β 2, 2 β 4, α7)、 人类神经型 nAChRs 亚型 ( 9 ΐθ, 6/ 3β2β3, α 7 ) 、 大鼠 α 9 α 10 nAChR的各种突变体， 以及小鼠和人类肌肉型 nAChRs ( (Χ ΐ β ΐ δ ε )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。含有 Ba2+的 ND-96緩冲液是用等摩尔浓度的 BaCl2 代替 CaCl2。 制得的样品用于例如下面的实施例 5-10。 实施例 5 : o O-芋螺毒素 GeXIVA 三种异构体阻断各种大鼠 nAChRs的实验
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 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)可以在淮注毒素或乙醜 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 ND96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 ( 2000 )位置时进行在线记录。 用 1mm外径 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 KC1 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 M，其他的亚型都为 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， 33 nM a O GeXIVA12 (实施例 3制备 )完全阻断了 由 Ach门控的 a 9 a 10 nAChR 开放产生的电流，且洗脱很快， 阻断是 可逆的（图 2A )。 3个异构体对 a 9 (X 10 nAChR均有很强的阻断活性， 在这 3个异构体中， a O GeXIVA12的活性最强， oc O GeXIVA14的 活性次之， ocO-GeXIVA13的活性相对最弱（图 2B，C，D )。 它们的半 阻断剂量 IC5Q和误差范围分别为： GeXIVA12， 4.6 nM (3.18-6.65 nM); GeXIVA13, 22.7 nM (11.8-43.5 nM); GeXIVA14, 7 nM (3.6-13.4 nM). 3 种异构体剂量反应曲线的斜率 （Hillslope)及误差范围分别为： GeXIVA12, 0.56(0.44-0.69); GeXIVA13, 0.78(0.29-1.26), GeXIVA14, 0.79(0.23-1.36). a 0-GeXIVA12 对各种 nAChRs 亚型均有不同程度的 阻断活性， 其半阻断剂量 IC5。和剂量反应曲线的斜率如表 2所示。
表 2: O GeXIVA12 对各种 nAChRs亚型的半阻断剂量 IC50和 剂量反应曲线的斜率
Figure imgf000032_0001
a O GeXIVA12对 α*β4 与 α*β2 nAChRs的阻断活性差异较 大， 对（X * β 2 nAChRs的阻断活性远高于对 α * β 4 nAChRs (图 3， A 一 E)； 对小鼠肌肉型 nAChR (Μαΐβΐδ ε )与 oc 7 nAChR亚型的 阻断活性较接近。 ocO-GeXIVA12更偏爱阻断含有 β 2 的 nAChRs, 包括 6/ 3β2β3, 4β2, α3β2 与 α2β2。 oc O GeXIVA12 对 9 10 nAChR 的阻断活性至少比其他亚型强 56-663 倍。 在小于 200nM低浓度下， oc O GeXIVA12是 oc 9 oc 10 nAChR的特异阻断剂， 对其他 nAChRs 亚型的阻断活性艮弱， 或几乎没有。 oc O-GeXIVA12 与其他超家族芋螺毒素的作用靶点生物活性比较列于表 3。
表 3: αΟ-GeXIVA与其他有关芋螺毒素的特性比较 毒素 ft g称 来源种类 序列 点 分子量 a9al0 nAChR »
C. TCRSSGRYCRSPYDR α*β2 nAChRs »
aO-GeXIVA 3452
generalis RRRYCRRITDACVA α*β4 nAChRs
nAChRs> NMDAR
CKGKGAKC SRLMYD Cav2.2 >Cav2.1
ω-MVIIA C. magus 2637
CCTGSCRSGKC# N> P/Q
Kvl.6>l.l> 1.2
pll4a
-1.3 -1.4 - (KJ-PIXIVA) a FPRPRICNLACRAGIG
1.5-2.1-3.4 2909 planorbis HKYPFCHCR#
pll4a (aJ- Muscle>a3p2
P1XIVA) nAChRs
ltl4a (aL-
C. litteratus MCPPLCKPSCTNC# Neuronal nAChR 1391 LtXIVA)
vill4a a GGLGRCIYNCMNSGG
N.D. 2871 (KL-VIIXIVA) Yillepinii GLSFIQCKTMCYA
a
HOOCCLYGKCRRYO
ψ-ΡΙΙΙΕ purpurasce Muscle nAChR 2715
GCSSASCCQR#
ns
GCCARAACAGIHQEL
a-LtIA C. litteratus α3β2 >α6/α3β2β3 1600
C#
GCCGPYONAACHOC
a
aA-EIVA GCKVGROOYCDROS Muscle nAChR 3094
ermineus
GG#
TYGIYDAKPOFSCAG
aC-PrXA C. partus LRGGCVLPONLROKF Muscle nAChR 3539
KE# DDySyCIINTRDSPWG
RCCRTRMCGSMCCP
aD-VxXXB C. vexillum α7 >α3β2 5735
RNGCTCVYHWRRGH GCSCPG (dimer)
KCNFDKCKGTGVYN CG(Gla)SCSC(Gla)GLH Muscle nAChR
aS-RVIIIA C.radiatus 5167
SCRCTYNIGSMKSGC Neuronal nAChR
ACICTYY
已知 oc 9 (x l0 nAChR对钙离子（Ca++ )具有很高的通透性。 通过 nAChRs 的钙离子内流可激活产生氯离子 （CD外流电流， 在非洲爪 蟾卵母细胞上， 这种电流占观察到的 oc 9 (x l0nAChR开放电流的 90% 以上。相反， 与钙离子接近的钡离子（Ba++ )不会激活产生氯离子电流。 因而， 我们用钡离子 ND96灌流液（Ba++ -ND96, 1.8 mM BaCl2代 替 CaCl2 )代替常规 ND96灌流液， 观察到的 α 9 oc lOnAChR开放电 流比用常规 ND96灌流液的电流小了很多， 这与以往的研究一致。 在 钡离子 ND96灌流液条件下， GeXIVA12对 oc 9 oc lOnAChR的阻断活性 最强， GeXIVA14次之， GeXIVA13活性相对最低（图 4 ) 。 具有 Ι-Π; III-IV二疏键连接方式的异构体 a 0-GeXIVA12对 oc 9 oc 10 nAChR的半 阻断剂量 （IC50 ) 及误差范围为 3.8 nM (3.1- 4.8 nM) , 曲线斜率
( Hillslope, nH )及误差范围为 0.71 (0.58-0.84); 具有 I-III; II-IV二疏 键连接方式的异构体 oc O-GeXIVA13对 oc 9 (x l0 nAChR的半阻断剂量
( IC50 )及误差范围为 37 nM (25.0-55.7 nM), 曲线斜率（ Hillslope )及 误差范围为 0.54 (0.42-0.65)； 具有 I-IV; II-III二硫键连接方式的异构体 a O GeXIVA14对 oc 9 oc 10 nAChR的半阻断剂量 ( IC5。 )及误差范围为 5.8 nM (4.7-7.1 nM) , 曲线斜率 （ Hillslope ) 及误差范围为 0.65 (0.56-0.73)。 在 ¾离子 ND96灌流液条件下， a O-GeXIVA的 3个异构 体的活性与在含有钙离子的正常 ND96 灌流液条件下的结果相似， 且 在钡离子 -ND96下， oc O-GeXIVA12与 oc 0-GeXIVA14的活性比在含 有钙离子的正常 ND96下更强一点， ¾离子 -ND96下的 oc 0-GeXIVA13 的活性却要比正常 ND96下弱一些。 因此， GeXIVA三种异构体的确是 阻断了 oc 9 (x l0nAChR， 而不是阻断因钙离子激活的氯离子电流。 实施例 6: 0-芋螺毒素 GeXIVA三种异构体阻断大鼠 NMDA受 体的实验
参照文献 ( Twede, V. D. et al. Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor. Biochemistry, 2009， 48: 4063-4073 )的方法， 采用与 nAChRs在非洲 l卵母细胞上表达类似的方法， 制备大鼠 NMDA受 体 4种亚型 NRl-2b /NR2A, NRl-2b /NR2B, NRl-2b /NR2C, NRl-2b /NR2D的相应 cRNA， 其浓度用 UV 260 nm下的 OD值进行测算。 解 剖收集非洲爪塘 ( 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 5 ng cRNA。 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 - 5天内用于 NMDA受 体的电压钳记录。用与 nAChRs相同的记录方式进行电压钳记录 NMDA 受体的电流， 只是所用的灌流液是不含镁离子的 ND96灌流液 ( Mg2+-free ND96 buffer )，其组成为 96.0 mM NaCl, 2.0 mM KC1， 1.8 mM CaC12， 5 mMHEPES (pH 7.2-7.5)。 因为 Mg2+在 -70 mV钳制电压 下，会阻断 NMDA受体。 NMDA受体激动剂溶液是含有终浓度为 200 μ Μ的谷氨酸（ glutamate 20 μ M甘氨酸（ glycine )的 Mg2+-free ND96„ a O GeXIVA12对 NR2C NMDAR亚型的阻断活性最强，洗脱较快。 O GeXIVA12对 4种 NMDA受体亚型的半阻断剂量（ IC5。 )及误差范 围分别为： NR2C，0.66 μ Μ (0.38-1.1 μ M); NR2B, 4.0 μ Μ (2.2-7.3 μ Μ)； NR2A, 3.7 μ Μ (2.8-5.0 μ M); NR2D, 5.2 μ Μ(1.7-15.7 μ M); O GeXIVA12对 4种 NMDA受体亚型剂量反应曲线斜率（ Hillslope ) 及误差范围分别为： NR2C， 0.13 (0.42-0.97); NR2B, 0.22(0.36-1.3); NR2A 0.10 (0.61-1.07); NR2D, 0.15 (0.10-0.78) (图 5 ) . a O-GeXIVA三种异 构体对各种 MDARs亚型的半阻断剂量 IC5。和剂量反应曲线的斜率列 于表 4。 O-GeXIVA三种异构体对
各种 NMDARs亚型的半阻断剂量 IC5。和剂量反应曲线的斜率
Figure imgf000036_0001
a O GeXIVA12对 4种 NMDA受体亚型的活性由强到弱的顺序为： NR2C >NR2A>NR2B>NR2D。 oc O GeXIVA14对 2种 NMDA受体 亚型 NR2B与 NR2A有较弱的阻断活性， 而 oc 0-GeXIVA13对 4种 NMDA受体亚型的阻断活性艮弱或没有。 实施例 7: o O-芋螺毒素 GeXIVA 三种异构体阻断其它的大鼠 nAChRs和小鼠肌肉型 nAChRs的实验
采用与实施例 4 和 5 相同的实验方法， 研究了 ocO-芋螺毒素 GeXIVA三种异构体阻断其它的大鼠 nAChRs的和小鼠肌肉型 nAChRs 作用。 3种异构体 ocO-GeXIVA12、 oc 0-GeXIVA13与 oc 0-GeXIVA14 对 nAChR受体其它各种亚型的浓度剂量反应曲线如图 6 ( A - J )所示。 总的来说， 3 种异构体对 oc*p2 nAChRs 的阻断活性高于对 oc * p 4 nAChRs (图 6 )； 3种异构体对小鼠肌肉型 nAChR (Μ ΐβΐδ ε ) 的阻断活性较接近， 它们的半阻断剂量 IC5()分别为： ocO-GeXIVA12， 394 nM; O-GeXIVA13, 671 nM; O-GeXIVA14, 473 nM.但对于 与 oc7 nAChR 亚型的阻断活性， 3 个异构体之间差异 大， 其 IC50 活性顺序为 ocO-GeXIVA12， 415nM > O-GeXIVA14, 1740 nM > a O GeXIVA13 ， 4960 nM. a O GeXIVA12 > GeXIVA14 > GeXIVA13这种活性顺序也出现在小鼠肌肉型 nAChR与神经型 a 3 p 2 nAChR的活性顺序上， 但这 3种异构体对 Μαΐβΐδ ε和 α3β2 nAChRs的阻断活性彼此之间差异很小。
可是对于 a 2 β 2， （Χ2β 4， α4β2， a 4 β 4 nAChRs的阻断活性 来说， GeXIVA14的阻断活性是最强的，对 a 2 p 2的 IC5。仅为 122 nM, 对 α4β2的 IC5。为 200nM。 对于 α3β4， a 6/ a 3 β 4 nAChR的阻断 活性来说， GeXIVA13 的阻断活性是最强的， 其 IC5。为 483 nM; GeXIVA14的阻断活性次之， IC5。为 611 nM; GeXIVA12的阻断活 性最弱， 其 IC5。为 806 nM。 不过 3种异构体对其他所有的 nAChRs亚 型的阻断活性都远小于对 a 9 a 10 nAChR的阻断活性。 3种异构体对不 同的 nAChRs亚型的差异性阻断， 可为进一步以 GeXIVA为模版， 设 计改造出一系列针对不同亚型的选择性阻断剂突变体提供了理论基 础。 实施例 8: o O-芋螺毒素 GeXIVA三种异构体阻断人类 nAChRs 的实验
采用与实施例 4 和 5 相同的实验方法， 研究了 ocO-芋螺毒素 GeXIVA 三种异构体阻断人类 nAChRs 的作用。 3 个异构体中， a O GeXIVA12和 GeXIVA14对人类 9a 10 nAChR均有艮强的阻断 活性， ocO-GeXIVA12的活性最强， a 0-GeXIVA14的活性次之， a O GeXIVA13的活性相对最弱（图 7， A - F )。它们的半阻断剂量 IC50 和误差范围分别为： GeXIVA12， 20 nM (12.4-33.2nM); GeXIVA13, 116 nM (65.4-204 nM); GeXIVA14, 47 nM (29.7-75.3 nM). 3种异构 体剂量反应曲线的斜率 （ Hillslope )及误差范围分别为： GeXIVA12， 0.91(0.49-1.32); GeXIVA13, 0.73(0.45-1.01), GeXIVA14, 0.67(0.46-0.88). 3种异构体对人类肌肉型 nAChR ( Human ΐβΐδ ε ) 的阻断活性较接近， 它们的半阻断剂量 1< 5()分别为： a O GeXIVA12, 497 nM; O-GeXIVA13, 485 nM; O-GeXIVA14, 365 nM.但对于与 a 7 nAChR 亚型的阻断活性， 3个异构体之间差异 4艮大，其 IC5。活性顺序为 a O GeXIVA12, 555 nM > a O GeXIVA14, 865 nM > a O GeXIVA13, 3300 nM. 对人类 a 9 a 10与 a 7 nAChRs 的阻断活性顺序 O GeXIVA12 > GeXIVA14 > GeXIVA13 与对大鼠 a 9 a 10与 a 7 nAChRs的阻断活性顺序一致。但这 3种异构体对人类 6/ 3 β 2 β 3 nAChR 的阻断活性顺序是 a O GeXIVA13≥ GeXIVA14 > GeXIVA12， 它们的半阻断剂量 IC5。 分别为： O GeXIVA13, 141 nM; O-GeXIVA14, 197 nM; O-GeXIVA12, 505 nM。 3种异构体对不同的人类 nAChRs亚型的差异性阻断， 有利 于设计改造出一系列 a O-GeXIVA类似物， 获得对不同亚型的选择性 阻断剂。 实施例 9: o O-芋螺毒素 GeXIVA三种异构体阻断大鼠 o 9o l0 nAChR受体的各种突变型的实验
将芋螺毒素与野生型 a 9 a 10 nAChR受体相结合部位的可能的关 键氛基酸进行点突变， 即单个氛基酸改变，制备 a 9 a 10 nAChR受体的 各种突变型， 突变位点在图 8中标明， 例如， "roc9R71G(xlO，，表示大 鼠（r)野生型 a 9 a 10 nAChR受体的 a 9亚基上， 第 71位的精氨酸 R(Arg) 突变为甘氛酸 G(Gly),其余突变体的表示方法以此类推。受体突 变体的制作是通过 PCR方法， 突变野生型 a 9 a 10 nAChR受体的 a 9 或 a 10亚基基因上，对应编码点突变氛基酸的密码子来实现的。所获突 变型， 按照与实施例 4 和 5相同的实验方法， 研究了 ocO-芋螺毒素 GeXIVA三种异构体阻断各种 a 9 a 10 nAChRs突变型的作用 （图 8 ) 。
测试了 3 种异构体 a O GeXIVA12、 a O GeXIVA13 与 a 0-GeXIVA14对 11个大鼠 oc9(xl0 nAChR受体的突变型的阻断活性。 其浓度剂量反应曲线如图 8 (A-K)所示。 总的趋势是， 3种异构体对 9 10 nAChR 的各种突变型均有艮强的阻断活性， O GeXIVA12 与 oc 0-GeXIVA14 对各种突变型的阻断活性接近， 且都比 α O GeXIVA13的活性要强， 这与对野生型 α 9 α 10 nAChR的阻断活性 与顺序一致。 3 种异构体的阻断活性顺序为 oc 0-GeXIVA12≥ GeXIVA14 > GeXIVA13。 活性变化较大的突变型有 5 个， a O GeXIVA12的半阻断剂量 IC5。在 46-59nM之间； a O GeXIVA14的 半阻断剂量 IC5。在 34-96nM之间； O GeXIVA13的半阻断剂量 IC50 在 106-232nM之间。 3种异构体对这 5个 (X 9 (X 10 nAChR突变型的半阻 断剂量 IC5。分别为： （l)roc9R71G(xlO， O-GeXIVA12, 59 nM; a O GeXIVA13, 232 nM; O-GeXIVA14, 61 nM。 （2) 9S14N 10, a O GeXIVA12， 53 nM; a O GeXIVA13， 108 nM; a O GeXIVA14, 60 nM。 （3) 9A24K lO, O-GeXIVA12, 52 nM;
O-GeXIVA13, 170 nM; O-GeXIVA14, 96 nM。 （4) 9E192Q a 10， a O GeXIVA12， 50 nM; a O GeXIVA13， 143 nM; a O GeXIVA14, 63 nM。 （5) 9S136N lO, O-GeXIVA12, 46 nM;
O-GeXIVA13, 106 nM; O-GeXIVA14, 34 nM。
活性变化很小的突变型有 6个， a O GeXIVA12的半阻断剂量 IC5。 在 14-32nM之间； a O GeXIVA14的半阻断剂量 IC50在 24-55nM之 间； ocO-GeXIVA13 的半阻断剂量 IC5。在 68-182nM之间。 对于 roc 9S136NoclO与 r a 9 a 10E56S两个突变型来说， 3种异构体的阻断活性 顺序为 a O GeXIVA14≥ GeXIVA12 > GeXIVA13。 对于 r oc9S136N(X 10突变型， ocO-GeXIVA14(IC5。， 34 nM)表现出比 GeXIVA12 ( IC50， 46 nM )更强的阻断活性。 3种异构体对这 6个 a 9 a 10 nAChR突变型 的半阻断剂量 IC5。分别为：（l)r a 9S117A a 10， a O GeXIVA12, 32 nM; a O GeXIVA13, 182 nM; a O GeXIVA14, 47 nM。（ 2 ) a a 10D116L, a O GeXIVA12, 28 nM; a O GeXIVA13, 122 nM; a O GeXIVA14, 45 nM。 （ 3 ) a 9 a 10E56S, a O GeXIVA12, 25 nM; a O GeXIVA13, 68 nM; a O GeXIVA14, 24 nM。 （ 4 ) a 9T56I a 10， a O GeXIVA12, 23 nM; O-GeXIVA13, 76 nM; O-GeXIVA14, 33 nM。 （ 5 ) a 9S6N 10, O-GeXIVA12, 20 nM; O-GeXIVA13, 119 nM; a O GeXIVA14, 55 nM。 (6) a 9T64I a 10, O-GeXIVA12, 14 nM;
O-GeXIVA13, 79 nM; O-GeXIVA14, 24 nM。
这些 a 9 a 10 nAChR突变型的突变位点， 是以往发现的 oc-芋螺毒 素与该受体相互结合的关键氨基酸（Ellison M， Feng ZP, Park AJ, Zhang X, Olivera BM, Mcintosh JM, Norton RS. Alpha-RglA, a novel conotoxin that blocks the alpha9alphal0 nAChR: structure and identification of key receptor-binding residues. J Mol Biol. 2008;377(4):1216-27 ) , a O-GeXIVA 的 3种异构体对这些突变型的活 性影响不大，活性变化是对野生型 a 9 a 10 nAChR的 10倍左右或以下。 说明 a O-GeXIVA与 a 9 a 10 nAChR的结合位点或部位与以往发现的 a-芋螺毒素的结合部位完全不同， 是新的作用位点。 实施例 10: o O-芋螺毒素 GeXIVA12特异阻断 o 9o l0 nAChR的 新颖位点
a O GeXIVA12阻断 a 9 a 10 nAChR的洗脱艮快（图 9A )。 a -CTX RglA [S4T; R9Cit; Y10Iodo，RllQ】 （简称 RglAM)是 a 9 a 10 nAChR 的特异阻断剂，但洗脱恢复非常慢（实验证实）， 也就是当毒素 RglAM 被洗脱后， 9 l0 nAChR回复至 ACh正常门控开放状态非常慢（图 9B) 。
根据二者洗脱速度的不同， 本发明人设计了竟争性实验（图 9C) 。 即先用 1 μΜ ocO-GeXIVA12温育阻断 oc9(xl0 nAChR lmin后， 再 用 20 nM a -CTX RglAM 与 1 μΜ a 0-GeXIVA12在同一蛙卵的细 胞槽中继续温育阻断 a 9 a 10 nAChR 5 min, Ach门控电流记录显示其 洗脱速度很慢， 且与单独用 20 nM -CTX RglAM的洗脱情形一样。 同时设置 ND96分别替换 a O GeXIVA12与 a -CTX RglAM, 作为正负 对照。 该结果表明， ocO-GeXIVA12不能阻止 oc-CTXRgIAM对 (X9(X 10 nAChR的阻断作用，证明两者结合 a 9 a 10 nAChR的位点完全不同， a 0-芋螺毒素 GeXIVA12结合的是 a 9 a 10 nAChR的新颖位点， 与以 前发现的 oc -芋螺毒素的结合位点不同， 且没有重叠。
研究表明， oc 9 (x l0 nAChR是治疗神经痛、 癌症化疗、 乳腺癌、 肺癌、 伤口愈合等的新 点 (Mcintosh, J. M.; Absalom, N.; Chebib, M.; Elgoyhen, A. Β·; Vincler, M.， Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochemical pharmacology 2009， 78 (7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vcl.l alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain research 2005, 1059 (2)， 149-58. Holtman, J. R.; Dwoskin, L. P.; Dowell, C; Wala, E. P.; Zhang, Ζ·; Crooks, P. A.; Mcintosh, J. M.， The novel small molecule alpha9alphal0 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic. European journal of pharmacology 2011， 670 (2-3), 500-8. Zheng, G.; Zhang, Ζ·; Dowell, C; Wala, E.; Dwoskin, L. P.; Holtman, J. R.; Mcintosh, J. Μ·; Crooks, P. A., Discovery of non-peptide, small molecule antagonists of alpha9alphal0 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain. Bioorganic & medicinal chemistry letters 2011， 21 (8), 2476-9. Chernyavsky, A. I.; Arredondo, J.; Vetter, D. E.; Grando, S. A., Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Experimental cell research 2007， 313 (16), 3542-55; Chikova, A.; Grando, S. A., Naturally occurring variants of human Alpha9 nicotinic receptor differentially affect bronchial cell proliferation and transformation. PloS one 2011, 6 (11), e27978. ) 。 因此， 本发明的 oc O-新超家族芋螺毒 素 GeXIVA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 11 : 重组 a 0-芋螺毒素 GeXIVAWT抑制 Sf9细胞生长的实 将野生型毒素 （ oc O-GeXIVAWT )基因插入大肠杆菌表达载体 pET22b (+) 的酶切位点 Nco I和 Xho I之间， 构建出 N端融合了 pelB leader, C端带有 His-tag纯化标签的融合蛋白表达载体。 按常规方法 进行 oc O-GeXIVAWT重组蛋白的分离纯化和制备。 通过 MTT法研究 了重组芋螺毒素 oc O-GeXIVAWT对 Sf9细胞（ Spodoptera frugiperda 9(Sf9) cells , 购自美国 Invitrogen公司）生长状况的影响 （图 10 ) 。 该 法是根据 MTT可透过细胞膜进入细胞内， 活细胞线粒体中的琥珀脱氢 酶能使外源性 MTT还原为难溶于水的蓝紫色的针状甲臜结晶并沉积在 细胞中， 而死细胞没有这种功能。 二甲亚砜（DMSO )能溶解沉积在细 胞中蓝紫色结晶物， 溶液颜色的深浅与所含的甲臜量成正比。 用酶标仪 在 570nm波长处测定其光吸收值（ OD值）， 可间接反映细胞数量。 实 验结果表明， 重组 oc O-GeXIVAWT能显著抑制 Sf9细胞的生长， 且存 在剂量效应， 在较高浓度下（ > 10 μ g/ml )可杀死 Sf9细胞。 Sf-9昆虫 细胞系来自农业害虫鳞翅目秋粘^ l ( Spodoptera Frugiperda )的卵巢 细胞系 Sf-21， 该昆虫细胞极易与作为生物农药的苜蓿加州核型多角体 病毒（AcMNPV杆状病毒）感染， 并且可以与所有杆状病毒表达载体 使用。 因而， 野生型重组芋螺毒素 oc O-GeXIVAWT在害虫控方面具有 艮好的应用潜力（Bruce C， Fitches EC, Chougule N, Bell HA, Gatehouse JA (2011) Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity. Toxicon 58:93-100·)。 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，其为或者包含一个或多个相同或者不同的选自如 下的 （1)至（3) 中任一项所述的氛基酸序列：
(1) SEQ ID NO: 7- 12中任一序列所示的氛基酸序列；
( 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) 所述序列有所 不同的赛基酸序列。
2. 根据权利要求 1所述的多肽， 其中， SEQIDNO: 12中的 N末端的第一个半胱氛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； 或者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氛酸形成 二硫键； 或者第一个半胱氨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； 所述多肽的羧基末端是 自由的 C-末端， 或者是酰胺化的。
3. 一种多核苷酸， 其编码权利要求 1或 2所述多肽的氛基酸 序列。
4. 根据权利要求 3所述的多核苷酸， 其为包含或者一个或多 个相同或不同的选自如下的（1)至（3)中任一项所述的核苷 ^列：
( 1 ) SEQ ID NO: 1-6中任一序列所示的核苷酸序列；
(2) SEQIDNO: 1-6中任一序列的互补序列； 或
(3)在严谊条件下能够与上述（1)或（2)中所述的核苷酸序列 杂交的核苷酸序列。
5. 一种核酸构建体，其包含权利要求 3或 4所述的多核苷酸。
6. 一种重组表达载体，其包含权利要求 5所述的核酸构建体； 具体地， 所述重组载体是重组 pET22b(+)载体。
7. 一种转化的细胞，其包含权利要求 6所述的重组表达载体； 具体地， 所述转化的细胞为转化的 Sf9细胞。
8. 一种融合蛋白， 其包含权利要求 1或 2所述的多肽。
9. 一种药物组合物， 其包含权利要求 1或 2所述的多肽， 或者 包含权利要求 8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。
10. —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或 2所述的多肽或者权利要 求 8所述的融合蛋白的步骤； 具体地， 所述乙酰胆碱受体是 a 9 a 10乙 酰胆碱受体。
11. 一种在体内或体外阻断 NMDA受体或者调节 NMDA受体 水平的方法， 包括使用有效量的权利要求 1或 2所述的多肽或者权利要 求 8所述的融合蛋白的步骤；具体地， 所述 NMDA受体是 NR2C NMDA 受体。
12. —种筛选乙酰胆碱受体抑制剂或确定乙酰胆碱受体亚型 的方法， 或者一种筛选 NMDA受体抑制剂或确定 NMDA受体亚型的方 法， 该方法包括： 在存在和不存在候选化合物存在的情况下将乙酰胆 碱受体或 NMDA受体与权利要求 1或 2所述的多肽或者权利要求 8所述 的融合蛋白进行接触的步骤； 具体地， 所述乙酰胆碱受体是 (X 9 (X 10 乙酰胆碱受体； 具体地， 所述 NMDA受体是 NR2C NMDA受体。
13. 权利要求 1或 2所述的多肽或者权利要求 8所述的融合蛋白 用于阻断乙酰胆碱受体或 NMDA受体的用途； 具体地， 所述乙酰胆碱 受体是（X 9 (X 10乙酰胆碱受体； 具体地， 所述 NMDA受体是 NR2C 匪 DA受体。
14. 权利要求 1或 2所述的多肽或者权利要求 8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或者在制备阻断 NMDA受体的药物或试剂中的用途； 具体地， 所述乙酰胆碱受体是 (X 9 (X 10乙酰胆碱受体； 具体地， 所述 NMDA受体是 NR2C NMDA受体。
15. 权利要求 1或 2所述的多肽或者权利要求 8所述的融合蛋白 在制备治疗和 /或预防神经系统疾病例如神经痛、 癌症化疗、 乳腺癌、 肺癌、 伤口愈合、 成瘾、 帕金森症、 瘫痫症、 局部缺血、 兴奋性神经 元细胞死亡或痴呆等的药物的用途， 或者用于制备杀灭害虫 （例如鳞 翅目秋粘夜蛾） 、 镇痛、 戒烟、 或戒毒的药物的用途； 具体地， 所述 神经痛由如下因素中的一种或多种导致： 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、 骨髄瘤、 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. 权利要求 1或 2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(Cys), But (Thr、 Ser、 Tyr),OBut (Asp),Boc (Lys); 半胱氛酸用 Trt或 Acm保护基团， 分别在 相应的半胱氨酸之间定点形成二硫键；
2 )将步骤 1 ) 中得到的线性多肽从树脂上切割下来， 并用冰乙醚 沉淀和洗涤回收线性多肽粗品，用制备型反向 HPLC C18柱 （ Vydac ) 纯化；
3 )将步骤 2 ) 中得到的产物进行两步氧化折叠。
17. 一种治疗和 /或预防和 /或辅助治疗神经系统疾病例如神经 痛、 乳腺癌、 肺癌、 成瘾、 瘫痫症、 局部缺血、 帕金森症、 痴呆、 或 兴奋性神经元细胞死亡等的方法， 或者一种杀灭害虫 （例如鳞翅目秋 粘夜蛾） 、 镇痛、 戒烟或戒毒的方法， 包括给予有效量的权利要求 1 或 2所述的多肽或者权利要求 8所述的融合蛋白的步骤； 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、 骨髄瘤、 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。
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