WO2016169028A1 - 三种芋螺毒素肽、其制备方法及应用 - Google Patents

三种芋螺毒素肽、其制备方法及应用 Download PDF

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WO2016169028A1
WO2016169028A1 PCT/CN2015/077302 CN2015077302W WO2016169028A1 WO 2016169028 A1 WO2016169028 A1 WO 2016169028A1 CN 2015077302 W CN2015077302 W CN 2015077302W WO 2016169028 A1 WO2016169028 A1 WO 2016169028A1
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cptx
conotoxin
conotoxin peptide
seq
polypeptide
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French (fr)
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刘杰
林志龙
闻博
童婷
莫芬
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深圳华大基因研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to the field of biomedical technology, in particular to three novel conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08, polynucleotides encoding the peptides, comprising the A polynucleotide construct, an expression vector and a host cell, a natural extraction and artificial synthesis method of the peptide, and a medical use of the peptide.
  • Ion channel is a kind of special hydrophilic protein micropores on the cell membrane. It is the material basis of the electrical activity of nerves and muscle cells. It is found that the function and structural abnormality of ion channels are related to the occurrence and development of many diseases. . Currently, about 15% of drugs use ion channels as targets. Among them, the voltage-dependent potassium channel (Kv) is the most diverse and widely distributed ion channel, which is divided into 12 families of Kv1-Kv12, and consists of four ⁇ subunits and four ⁇ subunits. The alpha subunit includes six transmembrane domains, pore-forming loops, and voltage sensors, and is symmetrically arranged around the central pore. The beta subunit or helper subunit interacts with the alpha subunit and can alter the properties of the channel complex, including but not limited to altering the electrophysiological or biophysical properties, expression levels or expression patterns of the channel.
  • Potassium channel drugs have potential therapeutic effects on angina, arrhythmia, hypertension, immunosuppression, and urinary incontinence, such as the third class of antiarrhythmic drugs amiodarone and bromobenzylamine, which inhibit voltage dependence on cardiomyocytes. Potassium channel to treat arrhythmias. Therefore, the development of specific potassium ion channel synergistic or antagonistic drugs is of great guiding significance for exploring the pathophysiological mechanisms of certain diseases, early diagnosis and discovery of specific therapeutic drugs.
  • the drugs that act on potassium channels in the clinic are mainly chemical drugs, while the drugs used in chemical drugs have large side effects. Compared with peptide drugs, the activity of peptide drugs is high, the toxicity is small, and the specificity is strong. Potassium ion channel drugs have broad application prospects.
  • Cone snails also known as “chicken heart snails”
  • Taxonomy Mollusca
  • Gastropoda Prosobranchia
  • Neogastropoda Neogastropoda
  • Conidae Conus.
  • China has discovered about 100 species of snails, mainly distributed in the Nansha Islands, the Xisha Islands, Hainan Island and the waters near Taiwan, and a few are distributed along the coasts of Guangdong and Guangxi.
  • Conopeptide (Conotoxin, CTX) is a class of biologically active peptide toxoids obtained from snails. It is mainly used for anesthesia, predation and defense competitors. Studies have shown that each snail may contain 1000 species. Conotoxin, that is, there are more than 500,000 conotoxins in the world. Conotoxin has the following characteristics: relatively low molecular weight, rich in disulfide bonds, stable structure, high activity, high selectivity and easy synthesis. They can efficiently and specifically act on acetylcholine receptors and other neurotransmitters. Various receptor subtypes.
  • conotoxin can selectively act on protein receptors such as ion channels, thereby affecting nerve conduction and producing different physiological effects.
  • ⁇ -Conotoxin which specifically blocks voltage-sensitive calcium channels, has great potential for neuromuscular probes in analgesia, neuroprotection, anticonvulsant, antitussive, etc. [5].
  • ⁇ -Conotoxin which specifically binds to various subtypes of sodium channels, alters its function.
  • the ⁇ -conotoxin PIIIA isoform blocks sodium channel Nav1.4 and is expected to be used in the treatment of cancer patients.
  • ⁇ -Conotoxin which specifically blocks voltage-sensitive potassium channels, plays an important role in cell proliferation.
  • ⁇ -conotoxins-PVIIA which stably regulates a potassium channel protein (HERG), has the potential to develop anticancer drug molecules. .
  • the analgesic drugs MrIA (Xen2174) and CVID (AM336) are in clinical stage I/II and II, respectively. In addition, some conotoxins are in preclinical research stage. ⁇ -Conotoxin SO3 and ⁇ -Conotoxin Ltl4a are currently used as analgesics. .
  • the study of the activity of conotoxin is important for the study of the structure and function of ion channels and the development of related drugs acting on ion channels or their lead compounds, and is also of great significance for the study of neurobiology.
  • the object of the present invention is to provide three novel conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08, a polynucleotide encoding the peptide, and a construct containing the polynucleotide , expression vectors and host cells, natural extraction and synthetic methods of the peptides, and medical uses of the peptides.
  • the present invention provides a conotoxin peptide ⁇ -CPTx-btl06 which is a polypeptide of the amino acid sequence of SEQ ID NO: 1; and wherein the amino acid sequence of the polypeptide contains three pairs of disulfide bonds This is a necessary condition for the conotoxin peptide to exert potassium channel inhibitory activity.
  • the present invention provides a conotoxin peptide ⁇ -CPTx-btl07 which is a polypeptide of the amino acid sequence shown as SEQ ID NO: 2; and wherein the amino acid sequence of the polypeptide contains two pairs of disulfide bonds This is a necessary condition for the conotoxin peptide to exert potassium channel inhibitory activity.
  • the present invention provides a conotoxin peptide ⁇ -CPTx-btl08 which is a polypeptide of the amino acid sequence shown as SEQ ID NO: 3; and wherein the amino acid sequence of the polypeptide contains two pairs of disulfide bonds This is a necessary condition for the conotoxin peptide to exert potassium channel inhibitory activity.
  • the inventors of the present invention extracted and identified the three conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention from the toxic tube of Conus betulinus in Hainan for the first time.
  • the amino acid sequence is: CRCEQTCGTCVPCC (as shown in SEQ ID NO: 1), VADDCCVDNVGTCCR (such as SEQ ID NO: 2) and ECCEDPPCRVNNMEHCPA (shown as SEQ ID NO: 3) have molecular weights of 1549.7 Da, 1568.7 Da, and 2043.3 Da, respectively.
  • the present invention provides a polynucleotide encoding the conotoxin peptide of any one of the first aspect to the third aspect.
  • the polynucleotide encoding the conotoxin peptide ⁇ -CPTx-btl06 of the amino acid sequence set forth in SEQ ID NO: 1 has the nucleotide sequence set forth in SEQ ID NO: 4, ie, its nucleoside The acid sequence is: TGCAGGGGCAGGCAGACTTGCGGAACATGCGTGCCGTGCTGC; the polynucleotide encoding the conotoxin peptide ⁇ -CPTx-btl07 encoding the amino acid sequence shown in SEQ ID NO: 2 has the nucleotide sequence shown in SEQ ID NO: 5, ie, its nucleus The nucleotide sequence is: GTGGCCGACGACTGCTGCGTAGACAATGTCGGCACTTGCTGCCGA; the polynucleotide encoding the conotoxin peptide ⁇ -CPTx-btl08 encoding the amino acid sequence of SEQ ID NO: 3 has the nucleotide
  • the invention provides a nucleic acid construct comprising the polynucleotide of the fourth aspect, and one or more control sequences operably linked thereto for directing production of the polypeptide in an expression host.
  • the present invention provides an expression vector comprising the nucleic acid construct of the fifth aspect.
  • the invention provides a transformed cell, wherein the nucleic acid construct of the fifth aspect or the expression vector of the sixth aspect is transformed.
  • the present invention provides the use of the conotoxin peptide according to any one of the first aspect to the third aspect, in the preparation of a medicament for inhibiting a potassium ion channel current; preferably, the potassium ion channel is voltage dependent Potassium ion channel.
  • DRG cells dorsal root ganglion cells
  • tetraaminopyridine (4-AP, which is a raw material for preparing cardiotonic, sterilizing, antiarrhythmic, antiulcer, and antispasmodic miraverine, is also a new antihypertensive drug, pinacidil.
  • the activity of the important raw materials was compared and analyzed to determine its inhibitory effect on potassium channels and its subsequent application.
  • the inventors have found through the above experiments that the three conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention have the function of specifically inhibiting voltage-dependent potassium ion channel current, and have A stronger inhibitory effect than the positive control, tetraaminopyridine.
  • the present invention provides the conotoxin peptide according to any one of the first aspect to the third aspect, wherein the preparation of the treatment of pain, epilepsy, stroke, spasm, muscle relaxation, Parkinson's disease, Alzheimer's disease, depression, Application in diseases of addiction, cardiovascular diseases, cancer, inflammation, etc.; preferably, the cardiovascular system diseases are hypertension, angina pectoris, coronary heart disease or arrhythmia.
  • the three conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07, and ⁇ -CPTx-btl08 of the present invention each have a function of specifically suppressing a voltage-dependent potassium ion channel current, It can be applied to the treatment of potassium ion channel related diseases, such as the above diseases.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conotoxin peptide of any one of the first aspect to the third aspect, and a pharmaceutically acceptable carrier.
  • the present invention provides the method for producing a conotoxin peptide according to any one of the first aspect to the third aspect, comprising:
  • the three conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention can also be obtained by extraction from natural organisms.
  • the inventors of the present invention extract the toxin polypeptide from the toxic tube of the genus Conus betulinus from the toxic tube of the genus Conus betulinus in Hainan, and then obtain the step of separation and identification.
  • the separation is carried out by strong cation exchange high performance liquid chromatography; preferably, mass spectrometry is used for peptide identification.
  • the conotoxin peptide having the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3 is extracted and identified using the following procedure:
  • the three conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention can be derived from natural active animal resources and belong to bioactive peptides, which are safer than traditional small molecule drugs.
  • the side effect is also smaller, and the selectivity is high and the specificity is strong; and because of its simple structure and easy artificial synthesis, the specific action on the potassium ion channel can effectively inhibit the potassium ion channel current and its activity is stronger than that of the tetraaminopyridine.
  • potassium ion channel related diseases for example, clinically for the treatment of pain, epilepsy, stroke, convulsions, muscle relaxation, Parkinson's disease, Alzheimer's disease, depression, addiction, cardiovascular disease, cancer, Diseases such as inflammation and other diseases have great potential.
  • Figure 1 shows the results of mass spectrometric sequence identification of the conotoxin peptide ⁇ -CPTx-btl06 of the present invention
  • Figure 2 shows the results of mass spectrometric sequence identification of the conotoxin peptide ⁇ -CPTx-btl07 of the present invention
  • Figure 3 shows the results of mass spectrometric sequence identification of the conotoxin peptide ⁇ -CPTx-btl08 of the present invention
  • Figure 4 shows the inhibition of potassium ion channel current by 10 ⁇ M conotoxin peptide ⁇ -CPTx-btl06;
  • Figure 5 shows the inhibition of potassium ion channel current by 10 ⁇ M conotoxin peptide ⁇ -CPTx-btl07;
  • Figure 6 shows the inhibition of potassium ion channel current by 10 ⁇ M conotoxin peptide ⁇ -CPTx-btl08;
  • Figure 7 shows the inhibition of potassium ion channel current by 5 mM 4-AP.
  • the conotoxin polypeptide treated as described above was enriched with a polypeptide in Strata-X C18 column snail venom.
  • Strata-X C18 enrichment operation was performed according to standard procedures: 1) 1 ml methanol was added to activate the column; 2) 1 ml 0.1% FA equilibrium column was added; 3) venom sample was loaded 1 ml, buffer (5% ACN + 0.1% FA) was washed, The wash was repeated 3 times; 4) 100% ACN elution, and the eluate was collected.
  • the enriched polypeptide was examined for molecular weight by MALDI-TOF-MS.
  • the LC/MS instrument is based on Shimadzu's nano HPLC chromatograph system and AB Sciex's Triple TOF 5600 mass spectrometer system.
  • Each of the pre-isolated polypeptide fractions was separated by a self-made 12 cm long, 75 ⁇ m inner diameter, and an integral capillary analysis column packed with a Welch Materials brand XB-C18 column having a pore size of 3 ⁇ m and a pore size of 300 nl/min.
  • the injection volume was detected to be 25 ⁇ l, and the elution gradient was that the concentration of liquid B was uniformly increased from 5% to 45% for 40 minutes.
  • the mass spectrometry collected electrospray voltage was 2.5 kV
  • the auxiliary gas pressure was 30 PSI
  • the sheath gas pressure was 15 PSI
  • the source temperature was 150 °C.
  • the acquisition of the first-order mass spectrum uses a high-resolution mode greater than or equal to 30,000.
  • the valence state of the parent ion is selected from the range of 2 to 5 charges.
  • 30 secondary mass spectrometry fragments can be continuously performed, thus completing 30 secondary spectral iones in 250 ms. The scan produces more than 120 secondary spectra per second, with a total cycle time of 3.3 seconds.
  • the mass spectral sequence search results corresponding to the conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention are shown in Fig. 1, Fig. 2 and Fig. 3, respectively. Shown.
  • the original mass spectrometry data detected by nanoLC-MS/MS was formatted into MGF, and then searched and identified by Mascot search software.
  • ⁇ -CPTx- of ⁇ -CPTx-btl06, VADDCCVDNVGTCCR shown as SEQ ID NO: 2 having a full-length amino acid sequence of CRCEQTCGTCVPCC (as shown in SEQ ID NO: 1) was selected by sequence characterization.
  • ⁇ -CPTx-btl08 of btl07 and ECCEDPPCRVNNMEHCPA shown as SEQ ID NO: 3).
  • the conotoxin linear peptide (completed by Shanghai Jill Biochemical Synthetic Co., Ltd.) was synthesized by the method of solid phase chemical synthesis of methoxycarbonyl (Fmoc), respectively, as shown in SEQ ID NO: 1, 2 or 3.
  • the polypeptide was dissolved in 0.1 M Tris-HCl, 0.1 M NaCl, 5 mM GSH, 0.5 mM GSSG, pH 7.4 at a mass to volume ratio of 1:10, and reacted at 25 ° C for 24 to 48 h.
  • the renaturation effect was examined by MALDI-TOF-MS.
  • Example 3 Inhibitory activity of the conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 of the present invention on potassium channel
  • the refolded conotoxin peptides ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 prepared in Example 2 were weighed, and the conotoxin peptide was detected by whole cell patch clamp method. Effects of ⁇ -CPTx-btl06, ⁇ -CPTx-btl07 and ⁇ -CPTx-btl08 on potassium channel of rat dorsal root ganglion (DRG) cells. 4-AP was used as a positive control.
  • the DRG cells in the incubator (from the dorsal root ganglion cells isolated from SD rats) were taken out, and the culture solution in the culture dish was replaced with the extracellular fluid at room temperature to prevent a drastic change in the temperature of the solution. Add extracellular fluid by gently adding it along the wall of the dish to prevent the cells from falling off the bottom of the dish. The cells with the changed liquid were placed under an inverted microscope to observe cells with smooth cell membrane and uniform cytoplasm, and patch clamp experiments were performed at room temperature of 20-25 °C.
  • a 100 ⁇ l borosilicate glass blank was selected as the glass microelectrode material.
  • the electrode tip port diameter is 1.5-3.0 ⁇ m by two-step drawing by the drawing device, and the initial resistance of the glass microelectrode after entering the liquid is 2-4 M ⁇ .
  • Electrode filling installation and moving into the liquid level After that, a positive positive pressure is applied to ensure that the tip of the electrode is clean, that is, the liquid junction potential is compensated. Under the inverted microscope, move the microelectrode over the selected cells and approach the cells, remove the positive pressure and start a little vacuum suction. After the high-impedance G ⁇ seal is formed between the electrode and the cell membrane, the electrode is performed. Fast capacitance compensation.
  • the cells were then clamped at -60 mV, giving a short, powerful negative pressure, breaking the cell membrane clamped in the microelectrode, and then compensating for slow capacitance. After the whole cell recording mode was formed, the cells were clamped to -90 mV, and after the cells were stable for 4-6 min, 60 mV depolarization stimulation was given, and the stimulation time was 400 ms, and the potassium channel current was recorded.
  • the peptide sample was added to the extracellular fluid to an effective concentration of 10 ⁇ M while observing changes in the potassium channel current (the experiment was repeated three times and the result was the average of three replicates).
  • the series resistance (Rs) is always constant within the range of ⁇ 10M ⁇ during the experiment, and the system series resistance (Rseries compensation) is compensated between 30% and 70%.
  • the present invention illustrates the product of the present invention, the detailed preparation process, and the use thereof by the above embodiments.
  • the present invention is not limited to the above detailed preparation process and use, that is, it does not mean that the present invention must be implemented by relying on the above detailed preparation process and use. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

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Abstract

本发明提供了三种新的芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08，其氨基酸序列分别如SEQ ID NO：1、2和3所示；还涉及编码这些肽的多核苷酸，含有所述多核苷酸的构建体、表达载体和转化的细胞，所述肽的制备方法以及在制备抑制钾离子通道电流的药物中的用途。

Description

三种芋螺毒素肽、其制备方法及应用 技术领域
本发明涉及生物医药技术领域，具体地，涉及三种新的芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08，编码所述肽的多核苷酸，含有所述多核苷酸的构建体、表达载体和宿主细胞，所述肽的天然提取及人工合成方法，以及所述肽的医药用途。
背景技术
离子通道(ion channel)是细胞膜上的一类特殊亲水性蛋白质微孔道，是神经、肌肉细胞电活动的物质基础，研究发现，离子通道的功能、结构异常与许多疾病的发生和发展有关。目前，大约有15％的药物以离子通道作为作用靶点。其中电压依赖性钾离子通道(Kv)是最丰富多样且分布广泛的离子通道，分为Kv1-Kv12共12个家族，由四个α亚基和四个β亚基构成多聚体。α亚基包括6个跨膜结构域、成孔环和电压传感器，且围绕中心孔对称排列。β亚基或辅助亚基与α亚基相互作用，并且可改变通道复合物的性能，包括但不限于改变通道的电生理学或生物物理学性能、表达水平或表达方式。
电压依赖性钾通道主要功能包括细胞兴奋性调节、神经递质释放、信号传导和平滑肌收缩等。钾通道药物对心绞痛、心律不齐、高血压、免疫抑制及尿失禁等疾病有潜在治疗作用，如第三类抗心律失常药物胺碘酮和溴苄胺，它们通过抑制心肌细胞上的电压依赖性钾通道来治疗心律失常。因此开发特异性钾离子通道协同或拮抗药物，对探讨某些疾病的病理生理机制、早期诊断及发现特异性治疗药物等均有十分重要的指导意义。但目前临床上作用于钾通道的药物主要以化学药物为主，而化学类药物用药的毒副作用较大，相比而言多肽类药物活性高、毒性反应较小、特异性强，开发成多种钾离子通道类药物有广阔的应用前景。
芋螺(Cone snails)，又称“鸡心螺”，据估计，全球约有500种芋螺，主要生长于热带海域，一般多生活在暖海，在生物分类学上属于软体动物门(Mollusca)，腹足纲(Gastropoda)，前鳃亚纲(Prosobranchia)，新腹足目(Neogastropoda)，芋螺科(Conidae)，芋螺属(Conus)。我国现今发现了约100余种芋螺，主要分布在南沙群岛、西沙群岛、海南岛及台湾附近海域，少数分布在广东、广西沿海。
芋螺毒素(Conopeptide，Conotoxin，CTX)是从芋螺中获得的一类具有生物活性的多肽类毒素，主要用于麻醉猎物、捕食和防御竞争对手，研究显示，每种芋螺可能含有1000种芋螺毒素，即全世界有可能有超过50万种芋螺毒素。芋螺毒素具有下述特点：相对分子量小、富含二硫键、结构稳定、高活性、高选择性及易于合成，它们能高效性、特异性地作用于乙酰胆碱受体及其他神经递质的各种受体亚型。
同样，芋螺毒素能选择性地作用于离子通道等蛋白受体，进而影响神经传导，产生不同的生理作用。例如，ω-Conotoxin，能专一阻断电压敏感型钙离子通道，作为神经生物学探针在镇痛、神经保护、抗惊厥、镇咳等方面具有巨大的应用潜力[5]。μ-Conotoxin，能与钠通道各种亚型特异结合，改变其功能，μ-conotoxin PIIIA异构体阻断钠通道Nav1.4，有望应用在癌症患者止痛治疗领域。κ-Conotoxin，特异性阻断电压敏感型钾离子通道，对细胞增殖有重要作用，κ-conotoxins-PVIIA，能稳定地制约一种钾通道蛋白(HERG)，具有开发成抗癌药物分子的潜力。
美国犹他大学教授B.M.Olivera的实验室是芋螺毒素研究的发源地，研究始于二十世纪七十年代。至今已分离得到的芋螺毒素有数千种，其中数十种已申请美国专利。它们在治疗慢性疼痛、癫痫、中风、痉挛、肌肉松弛、帕金森病、老年痴呆症、抑郁、成瘾、心血管疾病、癌症等疾病中发挥重要作用。目前，已有芋螺毒素多肽进入临床研究或经FDA批准作为新药使用，齐考诺肽(ziconotide)，Eisai公司研制，2004年获得美国FDA审批，2006年7月在英国上市，用于对其它治疗药物无应答或耐受不 良的严重慢性疼痛患者。镇痛药MrIA(Xen2174)和CVID(AM336)分别处于临床I/II、II阶段，另外有部分芋螺毒素处于临床前研究阶段，ω-Conotoxin SO3及α-Conotoxin Ltl4a目前作为镇痛药在研。
综上所述，芋螺毒素活性功能研究对于离子通道的结构和功能探讨以及研发作用于离子通道的相关药物或其先导化合物具有重要作用，对研究神经生物学也具有重要意义。
发明内容
本发明的目的在于提供三种新的芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08，编码所述肽的多核苷酸，含有所述多核苷酸的构建体、表达载体和宿主细胞，所述肽的天然提取及人工合成方法，以及所述肽的医药用途。
本发明通过以下技术方案实现上述目的：
第一方面，本发明提供了一种芋螺毒素肽κ-CPTx-btl06，其为如SEQ ID NO：1所示氨基酸序列的多肽；并且，所述多肽的氨基酸序列内含三对二硫键，这是该芋螺毒素肽发挥钾离子通道抑制活性的必要条件。
第二方面，本发明提供了一种芋螺毒素肽κ-CPTx-btl07，其为如SEQ ID NO：2所示氨基酸序列的多肽；并且，所述多肽的氨基酸序列内含两对二硫键，这是该芋螺毒素肽发挥钾离子通道抑制活性的必要条件。
第三方面，本发明提供了一种芋螺毒素肽κ-CPTx-btl08，其为如SEQ ID NO：3所示氨基酸序列的多肽；并且，所述多肽的氨基酸序列内含两对二硫键，这是该芋螺毒素肽发挥钾离子通道抑制活性的必要条件。
本发明的发明人首次从海南产桶形芋螺(Conus betulinus)毒管中提取并鉴定了本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08，其氨基酸分别序列为：CRCEQTCGTCVPCC(如SEQ ID NO：1所示)、VADDCCVDNVGTCCR(如 SEQ ID NO：2所示)和ECCEDPPCRVNNMEHCPA(如SEQ ID NO：3所示)，其分子量分别为1549.7Da、1568.7Da和2043.3Da。
第四方面，本发明提供了编码如第一方面至第三方面任一项所述的芋螺毒素肽的多核苷酸。
在具体实施方案中，编码如SEQ ID NO：1所示氨基酸序列的芋螺毒素肽κ-CPTx-btl06的多核苷酸具有如SEQ ID NO：4所示的核苷酸序列，即其核苷酸序列为：TGCAGGTGCGAGCAGACTTGCGGAACATGCGTGCCGTGCTGC；编码编码如SEQ ID NO：2所示氨基酸序列的芋螺毒素肽κ-CPTx-btl07的多核苷酸具有如SEQ ID NO：5所示的核苷酸序列，即其核苷酸序列为：GTGGCCGACGACTGCTGCGTAGACAATGTCGGCACTTGCTGCCGA；编码编码如SEQ ID NO：3所示氨基酸序列的芋螺毒素肽κ-CPTx-btl08的多核苷酸具有如SEQ ID NO：6所示的核苷酸序列，即其核苷酸序列为：GAATGCTGTGAGGATCCTCCTTGTCGGGTGAATAACATGGAGCATTGTCCCGCA。
第五方面，本发明提供了一种核酸构建体，其包含如第四方面所述的多核苷酸，以及与之可操作连接、可指导多肽在表达宿主中生产的一个或多个控制序列。
第六方面，本发明提供了一种表达载体，其包含如第五方面所述的核酸构建体。
第七方面，本发明提供了一种转化的细胞，其中转化了如第五方面所述的核酸构建体或如第六方面所述的表达载体。
第八方面，本发明提供了如第一方面至三方面任一项所述的芋螺毒素肽在制备抑制钾离子通道电流的药物中的应用；优选地，所述钾离子通道为电压依赖性钾离子通道。
发明人研究了本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08的生物学活性。具体地，采用全细胞膜片钳方法分别检测本发明的三种 芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08对背根神经节细胞(DRG细胞)离子通道的作用。实验中，钳制电压为-90mV，60mV去极化400ms，记录钾通道电流。加入样品后，记录样品对钾通道电流的影响。
通过与阳性对照物——四氨基吡啶(4-AP，是制备强心剂、灭菌剂、抗心律失常药、抗溃疡药及解痉药米尔维林的原料，也是新型降压药吡那地尔的重要原料)的活性进行比较分析，确定其对钾离子通道的抑制效果及其后续的应用方向。
发明人通过上述实验发现，本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08均具有特异性抑制电压依赖性钾离子通道电流的功能，并具有比阳性对照物——四氨基吡啶更强的抑制效果。
第九方面，本发明提供了如第一方面至第三方面任一项所述的芋螺毒素肽在制备治疗疼痛、癫痫、中风、痉挛、肌肉松弛、帕金森病、老年痴呆症、抑郁、成瘾、心血管系统疾病、癌症、炎症等疾病药物中的应用；优选地，所述心血管系统疾病为高血压、心绞痛、冠心病或心律失常。
根据第八方面所述，由于本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08均具有特异性抑制电压依赖性钾离子通道电流的功能，因而可应用于治疗钾离子通道相关疾病，比如上述各疾病。
第十方面，本发明提供了一种药物组合物，其包含如第一方面至第三方面任一项所述的芋螺毒素肽及药学上可接受的载体。
第十一方面，本发明提供了如第一方面至第三方面任一项所述的芋螺毒素肽的制备方法，其包括：
(1)采用固相化学合成法、优选芴甲氧羰基固相化学合成法，按如SEQ ID NO：1、2或3所示氨基酸序列合成芋螺毒素肽的线性肽；
(2)用谷胱甘肽法对步骤(1)所得线性肽进行氧化复性。
此外，本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08还可以通过从天然生物体中提取获得。具体地，本发明的发明人从海南桶形芋螺(Conus betulinus)的毒管中从海南桶形芋螺(Conus betulinus)的毒管中提取毒素多肽，然后进行分离、鉴定的步骤即可获得；优选地，采用强阳离子交换高效液相色谱进行分离；优选地，采用质谱法进行多肽鉴定。
在具体实施方案中，采用下述步骤提取并鉴定具有如SEQ ID NO：1、2或3所示氨基酸序列的芋螺毒素肽：
收集海南产桶形芋螺(Conus betulinus)，取毒管，提取其毒素多肽。经二硫苏糖醇(DTT)和碘乙酰胺(IAM)还原烷基化处理后，用强阳离子交换高效液相色谱(SCX-HPLC)分组分后采用纳升高效液相色谱-质谱联用仪(nanoLC-MS/MS)进行多肽质谱检测。产生的质谱数据经过数据解析和生物信息分析后得到芋螺毒素多肽完整的氨基酸序列。
有益效果
本发明的三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08可来源于天然活性动物资源，属于生物活性肽，与传统小分子药物相比，更为安全，副作用也更小，且选择性高、特异性强；又因其结构简单、易于人工合成，特异性作用于钾离子通道，能有效抑制钾离子通道电流且其活性比四氨基吡啶更强，因此可广泛用于钾离子通道相关疾病的治疗，例如，在临床上对于治疗疼痛、癫痫、中风、痉挛、肌肉松弛、帕金森病、老年痴呆症、抑郁、成瘾、心血管疾病、癌症、炎症等疾病治疗等疾病具有巨大潜力。
附图说明
图1显示本发明芋螺毒素肽κ-CPTx-btl06的质谱序列鉴定结果；
图2显示本发明芋螺毒素肽κ-CPTx-btl07的质谱序列鉴定结果；
图3显示本发明芋螺毒素肽κ-CPTx-btl08的质谱序列鉴定结果；
图4显示10μM芋螺毒素肽κ-CPTx-btl06对钾离子通道电流的抑制作用；
图5显示10μM芋螺毒素肽κ-CPTx-btl07对钾离子通道电流的抑制作用；
图6显示10μM芋螺毒素肽κ-CPTx-btl08对钾离子通道电流的抑制作用；
图7显示5mM 4-AP对钾离子通道电流的抑制作用。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1 本发明三种芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08的提取和鉴定
1、芋螺毒液提取及还原烷基化
将4只海南产桶形芋螺(Conus betulinus)砸壳后解剖剪取毒管，收集芋螺毒液。使用Bradford法测定毒液中蛋白浓度为6.48mg/ml。取总蛋白量为0.5mg，加入终浓度为1mM的DTT，于56℃反应1h，还原冷却至室温后加入终浓度为55mM的IAM于室温暗室反应45min。
2、毒素多肽富集
将经上述处理的芋螺毒素多肽，用Strata-X C18柱多芋螺毒液中多肽进行富集。Strata-X C18富集操作按标准规程操作：1)加1ml甲醇活化柱子；2)加1ml 0.1％FA平衡柱子；3)毒液样品上样1ml，buffer(5％ACN+0.1％FA)wash，重复wash 3次；4)100％ACN洗脱，收集洗脱液。富集多肽用MALDI-TOF-MS检测其分子量大小。
3、芋螺毒素多肽序列鉴定
将240μg混合肽经SCX-HPLC(Shimadzu)系统进行组分分离：bufferA：10mM KH2PO4in 25％ACN，pH 3.5；buffer B是在buffer A的基础上加入500mM氯化钾； 流速1ml/min，0-40％的线性二元梯度的buffer B洗脱10分钟，40-90％的buffer B洗脱2分钟，90％的buffer B洗脱3分钟，214nm进行吸光度检测，通过梯度洗脱共收集10个馏分。收集馏分经C18固相萃取柱除盐(Strata-X，Phenomenex)后用30μl的0.1％的甲酸复溶，进行nanoLC-MS/MS分析。
4、nanoLC-MS/MS分析
液质联用仪采用的是岛津的nano HPLC色谱仪系统和AB Sciex的Triple TOF 5600质谱仪系统。每个预分离好的多肽组分分别经过自制的12em长，75μm内径，填充了粒径3μm孔径120A的Welch Materials品牌XB-C18柱料的Ultimate毛细管分析柱分离，流速为300nl/min。检测进样体积为25μl，洗脱梯度为B液浓度从5％均匀上升到45％经过40min。质谱采集的电喷雾电压为2.5kV，辅气气压为30PSI，鞘气气压为15PSI，源温度为150℃。一级质谱的采集使用大于或等于30000的高分辨率模式。二级质谱的采集，选择母离子价态在2电荷到5电荷的范围，扫描一次一级质谱后可接着连续做30个二级质谱碎裂，这样在250ms内完成对30个二级谱子离子的扫描，每秒可产生超过120张二级谱，总的一个循环时间为3.3秒。
在得到的原始质谱数据中，相应于本发明芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08的质谱序列搜索比对结果分别如图1、图2和图3所示。
5、数据分析
将nanoLC-MS/MS检测得到的原始质谱数据进行格式转换成MGF后，再用Mascot搜索软件进行数据搜索鉴定。得到的多肽序列中，通过序列特征分析选取全长氨基酸序列为CRCEQTCGTCVPCC(如SEQ ID NO：1所示)的κ-CPTx-btl06、VADDCCVDNVGTCCR(如SEQ ID NO：2所示)的κ-CPTx-btl07和ECCEDPPCRVNNMEHCPA(如SEQ ID NO：3所示)的κ-CPTx-btl08。
实施例2 本发明芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08的化学 合成
应用芴甲氧羰基(Fmoc)固相化学合成的方法分别合成如SEQ ID NO：1、2或3所示序列的芋螺毒素线性肽(由上海吉尔生化合成公司完成)。
化学合成的多肽使用谷胱甘肽氧化复性法进行复性，即：
取多肽按质量体积比1∶10溶解于0.1M Tris-HCl，0.1M NaCl，5mM GSH，0.5mM GSSG，PH 7.4溶液中，25℃反应24～48h。用MALDI-TOF-MS检测复性效果。
实施例3 本发明芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08对钾离子通道的抑制活性
具体地，分别称取实施例2中制备的、复性后的芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08，通过全细胞膜片钳方法检测芋螺毒素肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08对大鼠背根神经节(DRG)细胞钾离子通道的作用。以4-AP为阳性对照。
膜片钳中细胞内、外液配置：
细胞外液：140mM NaCl，4mM KCl，1mM MgCl2，2mM CaCl2，5mM D-葡萄糖一水物(D-Glucose monohydrate)，10mM HEPES(pH＝7.4)；细胞内液：20mM KCl，110mM天冬氨酸钾(KAspartic)，1mM MgCl2，5mM EGTA，10mM HEPES(pH＝7.2)。
取出恒温培养箱中的DRG细胞(从SD大鼠中急性分离培养的背根神经节细胞)，用室温下平衡好的细胞外液更换培养皿内的培养液，以防止溶液温度的剧烈变化。加细胞外液是用枪沿着皿壁轻轻加入，以防止细胞从培养皿底部脱落。将换好液的细胞放置倒置显微镜下观察，选择细胞膜较为光滑、细胞质均匀的细胞，在室温20-25℃条件下进行膜片钳实验。
选用100μl硼硅酸盐玻璃毛坯为玻璃微电极材料。经拉制仪两步拉制使电极尖端口径为1.5-3.0μm，玻璃微电极入液后初始电阻为2-4MΩ。电极充灌安装并移入液面 后即给持续正压以保证电极尖端清洁，即进行液接电位补偿。在倒置显微镜下将微电极移至所选细胞上方并接近细胞，去除正压并开始稍施负压吸引，待电极与细胞膜之间形成高阻抗的吉欧(GΩ)封接后，即进行电极快电容(fast capacitance)补偿。然后将细胞钳制在-60mV，给予一短而有力的负压，将钳制在微电极中的细胞膜迅速打破，再进行细胞慢电容(Slow capacitance)补偿。形成全细胞记录模式后将细胞钳制为-90mV，细胞稳定4-6min后，给予60mV去极化刺激，刺激时间为400ms，记录钾通道电流。向细胞外液中加入多肽样品使其有效浓度达10μM同时观察记录钾通道电流的变化(实验重复三次，结果为三次重复的平均值)。串联电阻(Rs)在实验过程中在＜10MΩ的范围之内始终保持不变，系统串联电阻(Rseries compensation)补偿在30～70％之间。
多肽κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08的检测结果分别如图4、5和6所示，4-AP的检测结果如图7所示。图4-7结果显示，10μM的多肽样品：κ-CPTx-btl06、κ-CPTx-btl07和κ-CPTx-btl08对DRG神经元细胞钾离子通道电流的抑制率分别为25.78％、23.79％和24.58％，而阳性对照组5mM 4-AP对DRG神经元细胞钾离子通道电流的抑制率只有15.17％，比多肽样品较弱(数据如表1)。
表1、本发明三种芋螺毒素肽对钾离子通道抑制率的膜片钳检测结果
Figure PCTCN2015077302-appb-000001
申请人声明，本发明通过上述实施例来说明本发明的产品、详细制备工艺及其用 途，但本发明并不局限于上述详细制备工艺和用途，即不意味着本发明必须依赖上述详细制备工艺和用途才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (11)

  1. 一种芋螺毒素肽κ-CPTx-bt106，其为如SEQ ID NO：1所示氨基酸序列的多肽。
  2. 一种芋螺毒素肽κ-CPTx-bt107，其为如SEQ ID NO：2所示氨基酸序列的多肽。
  3. 一种芋螺毒素肽κ-CPTx-bt108，其为如SEQ ID NO：3所示氨基酸序列的多肽。
  4. 编码如权利要求1-3任一项所述芋螺毒素肽的多核苷酸。
  5. 一种核酸构建体，其包含如权利要求4所述的多核苷酸，以及与之可操作连接、可指导多肽在表达宿主中生产的一个或多个控制序列。
  6. 一种表达载体，其包含如权利要求5所述的核酸构建体。
  7. 一种转化的细胞，其中转化了如权利要求5所述的核酸构建体或如权利要求6所述的表达载体。
  8. 如权利要求1-3任一项所述的芋螺毒素肽在制备抑制钾离子通道电流的药物中的应用；优选地，所述钾离子通道为电压依赖性钾离子通道。
  9. 如权利要求1-3任一项所述的芋螺毒素肽在制备治疗疼痛、癫痫、中风、痉挛、肌肉松弛、帕金森病、老年痴呆症、抑郁、成瘾、心血管系统疾病、癌症或炎症的药物中的应用；优选地，所述心血管系统疾病为高血压、心绞痛、冠心病或心律失常。
  10. 一种药物组合物，其包含如权利要求1-3任一项所述的芋螺毒素肽及药学上可接受的载体。
  11. 如权利要求1-3任一项所述的芋螺毒素肽的制备方法，其包括：
    (1)采用固相化学合成法、优选芴甲氧羰基固相化学合成法，按如SEQ ID NO：1、2或3所示氨基酸序列合成芋螺毒素肽的线性肽；
    (2)用谷胱甘肽法对步骤(1)所得线性肽进行氧化复性。
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