WO2016101274A1 - 芋螺毒素肽κ-CPTx-btl01、其制备方法及应用 - Google Patents

芋螺毒素肽κ-CPTx-btl01、其制备方法及应用 Download PDF

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WO2016101274A1
WO2016101274A1 PCT/CN2014/095195 CN2014095195W WO2016101274A1 WO 2016101274 A1 WO2016101274 A1 WO 2016101274A1 CN 2014095195 W CN2014095195 W CN 2014095195W WO 2016101274 A1 WO2016101274 A1 WO 2016101274A1
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cptx
conotoxin peptide
peptide
btl01
substituted
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French (fr)
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刘杰
林志龙
闻博
童婷
莫芬
彭超
石琼
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深圳华大基因研究院
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Priority to EP14908842.9A priority Critical patent/EP3239167B1/en
Priority to CN201480083949.0A priority patent/CN107108695B/zh
Priority to PCT/CN2014/095195 priority patent/WO2016101274A1/zh
Priority to US15/539,709 priority patent/US10501498B2/en
Publication of WO2016101274A1 publication Critical patent/WO2016101274A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof

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  • the present invention relates to the field of biomedical technology, and in particular to a novel conotoxin peptide ⁇ -CPTx-btl01, a polynucleotide encoding the peptide, a construct containing the polynucleotide, an expression vector and a host cell, Natural extraction and synthetic methods of peptides, and medical uses of the peptides.
  • HVA calcium channels are transmembrane proteins that are widely distributed in excited cells, regulate extracellular Ca 2+ into the cytoplasm, and participate in a series of Ca 2+ -dependent physiological activities such as hormone and neurotransmitter release, muscle contraction and cell signaling. . According to the sensitivity to voltage, it is divided into low voltage activated calcium channel (LVA) or T-type calcium channel (Cav3.x) and high voltage activated calcium channel (HVA).
  • HVA calcium channel includes L-type (Cav1. x), P/Q-type (Cav2.1), N-type (Cav2.2) and R-type (Cav2.3) calcium channels.
  • the Cav1.x channel is involved in muscle contraction and cell secretion, and Cav2.x is involved in neurotransmitter release and neuronal excitation regulation.
  • the calcium channel consists of the predominantly functional alpha subunit and the auxiliary functional beta, gamma and delta subunits.
  • Calcium channel blockers are widely found in animal venoms, such as cockroaches, spiders, etc., and many drugs such as dihydropyridine small molecule calcium channel blockers have been developed. Typical drugs are selective calcium blockers nifedipine. These drugs are mainly used in the treatment of hypertension, coronary heart disease, arrhythmia, angina pectoris, cerebrovascular disease, etc., and are the most widely used and frequently used target drugs.
  • ziconotide is a class of N-type calcium channel blockers for the treatment of refractory chronic pain.
  • Polypeptide calcium channel blockers will become the hotspot of future calcium channel blocker drug development due to its many advantages, such as high selectivity, high lipophilicity and high safety.
  • Hypertension is one of the most common cardiovascular diseases in the world today and a major risk factor for cardiovascular and cerebrovascular diseases.
  • the results of the research published by the Center for Disease Control and Prevention of China CDC show that the number of patients with hypertension in China has exceeded 330 million.
  • calcium channel blockers are the main force in the current antihypertensive drug retail market, which mainly inhibits extracellular calcium influx by blocking calcium channel on the membrane of myocardial and vascular smooth muscle cells, and lowers intracellular calcium ion levels. It causes the smooth muscle contraction rate of cardiovascular and other tissues to decrease, and vasodilation, thereby lowering blood pressure.
  • the snail is a kind of toxic animal widely distributed in the tropical ocean. Its venom contains a large number of peptide ion channel active substances, such as ⁇ -Conotoxin, which can block the voltage-sensitive calcium channel and develop the calcium channel. An important natural resource for blockers.
  • the object of the present invention is to provide a novel conotoxin peptide ⁇ -CPTx-btl01, a polynucleotide encoding the same, a construct containing the polynucleotide, an expression vector and a transformed host cell, and the natural extraction of the peptide And synthetic methods, as well as the medical use of the peptide.
  • the present invention provides a conotoxin peptide ⁇ -CPTx-btl01 which is:
  • the amino acid sequence of the polypeptides (a) and (b) contains a three-pair disulfide bond, which is a necessary condition for the polypeptide to exert a calcium channel inhibitory activity, and thus, as in the amino acid sequence shown in SEQ ID NO: 1. Cysteine cannot be substituted, added or deleted.
  • the substitution of the one or more amino acids is selected from the group consisting of A group consisting of:
  • the inventors of the present invention extracted and identified the conotoxin peptide ⁇ -CPTx-btl01 of the present invention from the toxic tube of Conus betulinus in Hainan for the first time, and its amino acid sequence is: KCCTMSVCQPPPVCTCCA (ie SEQ ID NO: 1 ), its molecular weight is 1872.72 Daltons.
  • the inventors have also found that by carrying out a specific substitution of one or more specific amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, it is also possible to retain its original function, namely the polypeptide (b).
  • the invention provides a polynucleotide encoding the conotoxin peptide ⁇ -CPTx-btl01 as described in the first aspect.
  • the polynucleotide encoding the same has the nucleotide sequence set forth in SEQ ID NO: That is, the nucleotide sequence thereof is: AAGTGCTGTACTATGTCGGTATGCCAGCCGCCGCCAGTGTGTACATGCTGCGCT.
  • the invention provides a nucleic acid construct comprising the polynucleotide of the second aspect, and one or more control sequences operably linked thereto for directing production of the polypeptide in an expression host.
  • the invention provides an expression vector comprising the nucleic acid construct of the third aspect.
  • the invention provides a transformed cell, wherein the nucleic acid construct of the third aspect or the expression vector of the fourth aspect is transformed.
  • the present invention provides the use of the conotoxin peptide ⁇ -CPTx-btl01 according to the first aspect for the preparation of a medicament for inhibiting calcium ion channels; preferably, the calcium ion channel is a high voltage activated calcium ion aisle.
  • the calcium was determined by comparison with the positive control nifedipine (first-generation calcium channel blocker, antihypertensive, anti-angina drug, one of the world's best-selling drugs in the mid-1980s). Ion channel inhibition effect and its subsequent application direction.
  • positive control nifedipine first-generation calcium channel blocker, antihypertensive, anti-angina drug, one of the world's best-selling drugs in the mid-1980s.
  • the conotoxin peptide ⁇ -CPTx-btl01 of the present invention can effectively inhibit the high voltage activation of calcium channel (HVA) current, and the inhibitory effect is higher than that of the positive control nifedipine.
  • HVA calcium channel
  • the present invention provides the use of the conotoxin peptide ⁇ -CPTx-btl01 according to the first aspect in the preparation of a medicament for treating diseases of the cardiovascular system; preferably, the cardiovascular system disease is hypertension, angina pectoris Or coronary heart disease.
  • the conotoxin peptide ⁇ -CPTx-btl01 of the present invention can effectively inhibit high voltage activation of calcium channel (HVA) current, it can be applied to treat diseases such as hypertension, angina pectoris, and coronary heart disease.
  • HVA calcium channel
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conotoxin peptide ⁇ -CPTx-btl01 as described in the first aspect, and a pharmaceutically acceptable carrier.
  • the present invention provides the preparation method of the conotoxin peptide ⁇ -CPTx-btl01 according to the first aspect, which comprises:
  • conotoxin peptide ⁇ -CPTx-btl01 is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1, it can also be obtained by extraction from a natural organism. Specifically, the inventors extracted the toxin polypeptide from the toxic tube of the genus Conus betulinus in Hainan, and then obtained the steps of separation and identification; preferably Separation by strong cation exchange high performance liquid chromatography; preferably, mass spectrometry is used for peptide identification.
  • the conotoxin peptide ⁇ -CPTx-btl01 having the amino acid sequence set forth in SEQ ID NO: 1 is extracted and identified using the following procedure:
  • the conotoxin peptide ⁇ -CPTx-btl01 of the invention can be derived from natural active animal resources and belongs to a calcium channel blocker, and has the advantages of high selectivity, high lipophilicity and high safety compared with traditional small molecule drugs; Because of its simple structure and easy artificial synthesis, it can effectively inhibit calcium channel current and its activity is higher than that of nifedipine. Therefore, it can be widely used in the treatment of calcium ion channel related diseases, for example, in clinical treatment of hypertension and crown. Diseases such as heart disease and angina have great potential.
  • Figure 1 shows the results of mass spectrometric sequence identification of the conotoxin peptide ⁇ -CPTx-btl01 of the present invention
  • Figure 2 shows the inhibition of high voltage activated calcium channels by 10 ⁇ M conotoxin peptide ⁇ -CPTx-btl01;
  • Figure 3 shows the inhibition of high voltage activated calcium channels by 10 ⁇ M nifedipine.
  • 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 pre-separated peptide component was individually filled with a 12 cm long, 75 ⁇ m inner diameter.
  • the Welch Materials brand XB-C18 column with a particle size of 3 ⁇ m and a pore size of 120 A was separated by an Ultimate capillary analysis column at a flow rate 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 original mass spectrometry data detected by nanoLC-MS/MS was formatted into MGF, and then searched and identified by Mascot search software.
  • the ⁇ -CPTx-btl01 polypeptide having the full-length amino acid sequence of KCCTMSVCQPPPVCTCCA was selected by sequence characterization.
  • the conotoxin linear peptide of the sequence shown in SEQ ID NO: 1 was synthesized by the method of solid phase chemical synthesis of methoxycarbonyl (Fmoc) (completed by Shanghai Jill Biochemical Co., Ltd.).
  • 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 peptide ⁇ -CPTx-btl01 of the present invention on calcium channel
  • the inhibitory activity of the conotoxin peptide ⁇ -CPTx-btl01 of the present invention on calcium channel was examined using a patch clamp technique. Specifically, the refolded conotoxin peptide ⁇ -CPTx-btl01 prepared in Example 2 was weighed, and the conotoxin peptide ⁇ -CPTx-btl01 was detected by whole cell patch clamp method on the rat dorsal root ganglia ( DRG) cell ion channel use. Nifedipine 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 ⁇ .
  • a continuous positive pressure is applied to ensure that the electrode tip 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.
  • the cells After forming the whole cell recording mode, the cells were clamped to -90 mV, and after the cells were stable for 4-6 min, the cells were given two depolarizing stimuli, the first one was from -90 mV to -30 mV, the stimulation was 250 ms, and the second was from -60 mV to 0 mV.
  • the stimulation was 250 ms, the interval between the two stimuli was 500 ms, and the LVA calcium channel and the HVA calcium channel were recorded.
  • the peptide sample was added to the extracellular fluid to an effective concentration of 10 ⁇ M while the change in calcium channel current was observed (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 results of the conotoxin peptide ⁇ -CPTx-btl01 are shown in Figure 2.
  • the detection results of nifedipine are shown in Figure 3.
  • the inhibition rate of 10 ⁇ M ⁇ -CPTx-btl01 on HVA calcium channel current in DRG neurons 52.17% (as shown in Table 1).
  • the above results show that the same concentration of ⁇ -CPTx-btl01 has higher inhibition rate of HVA calcium channel current than nifedipine, suggesting that it can be used as a drug candidate for antihypertensive drugs.

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Abstract

提供了一种新的芋螺毒素肽κ-CPTx-btl01，其具有SEQ ID NO：1所示氨基酸序列或者为如SEQ ID NO：1所示氨基酸序列经过一个或多个氨基酸取代、添加或缺失而形成的、保留原有功能的衍生多肽；还提供了编码该肽的多核苷酸，含有该多核苷酸的构建体、表达载体和转化的细胞，该肽的制备方法及用于钙离子通道相关疾病的治疗的用途。

Description

芋螺毒素肽κ-CPTx-btl01、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，涉及一种新的芋螺毒素肽κ-CPTx-btl01，编码该肽的多核苷酸，含有该多核苷酸的构建体、表达载体和宿主细胞，该肽的天然提取及人工合成方法，以及该肽的医药用途。
背景技术
钙离子通道是广泛分布于兴奋细胞的跨膜蛋白，调节细胞外的Ca2+进入细胞质，参与一系列Ca2+依赖的生理活动，如激素及神经递质释放、肌肉收缩和细胞信号传导等。根据对电压的敏感性不同分为低电压激活钙离子通道(LVA)或T-型钙通道(Cav3.x)和高电压激活钙离子通道(HVA)，HVA钙通道包括L-型(Cav1.x)、P/Q-型(Cav2.1)、N-型(Cav2.2)和R-型(Cav2.3)钙通道。Cav1.x通道参与肌肉收缩和细胞分泌，Cav2.x参与神经递质释放和神经元兴奋调节。钙通道由起主要功能的α亚基和辅助功能的β、γ及δ亚基组成。
钙通道阻滞剂广泛存在于动物毒液中，如蜈蚣、蜘蛛等，目前已开发出很多如二氢吡啶类小分子钙通道阻滞剂药物，典型药物有选择性钙离子阻滞剂硝苯地平，该类药物在临床上主要应用于治疗高血压、冠心病、心律失常、心绞痛、脑血管病等，属当前应用最广泛和频率较高的一类靶点药物。
然而，目前已开发的多肽类钙通道阻滞剂药物则相对较少，如齐考诺肽是一类N-型钙通道阻滞剂，用于治疗难治性慢性疼痛。多肽类钙通道阻滞剂因其众多优势，如高选择性、高亲脂性和高安全性，将成为未来钙通道阻滞剂药物开发的热点。
高血压是当今世界最常见的心血管疾病之一，也是心脑血管疾病的主要危险因素。中国疾控中心慢病防控中心公布的研究结果显示，我国高血压患者人数已突破3.3亿 人，平均每3个成人中就有1人是高血压患者。一线抗高血压药物有5类，分别是钙通道阻滞剂(CCB)、血管紧张素转换酶抑制剂(ACE I)、血管紧张素受体阻滞剂(ARB)、利尿剂和β受体阻滞剂。其中，钙通道阻滞剂是目前降压药零售市场的主力军，其主要通过阻断心肌和血管平滑肌细胞膜上的钙离子通道，抑制细胞外的钙离子内流，使细胞内钙离子水平降低而引起心血管等组织平滑肌收缩率降低，血管舒张，从而降低血压。
芋螺是一类广泛分布于热带海洋的有毒动物，其毒液中含有大量的多肽类离子通道活性物质，例如ω-Conotoxin，能专一阻断电压敏感型钙离子通道，是开发多肽类钙通道阻滞剂类降压药的重要天然资源。
发明内容
本发明的目的在于提供一种新的芋螺毒素肽κ-CPTx-btl01，编码该肽的多核苷酸，含有该多核苷酸的构建体、表达载体和转化的宿主细胞，该肽的天然提取及人工合成方法，以及该肽的医药用途。
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-btl01，其为：
(a)如SEQ ID NO：1所示氨基酸序列的多肽；
(b)如SEQ ID NO：1所示氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的，具有(a)多肽功能的由(a)衍生的多肽；所述被取代、添加或缺失的氨基酸中不包括半胱氨酸；所述功能为抑制钙离子通道的活性；
所述(a)和(b)多肽的氨基酸序列内含三对二硫键，其为所述多肽发挥钙离子通道抑制活性的必要条件，因此，如SEQ ID NO：1所示氨基酸序列中的半胱氨酸是不能被取代、添加或缺失的。
上述芋螺毒素肽κ-CPTx-btl01中，作为优选，所述一个或多个氨基酸的取代选自 由以下组成的组：
(i)第1位的赖氨酸被精氨酸取代；
(ii)第4位的苏氨酸被丝氨酸取代；
(iii)第7位的缬氨酸被亮氨酸或异亮氨酸取代；
(iv)第9位的谷氨酰胺被天冬酰胺取代；
(v)第13位的缬氨酸被亮氨酸或异亮氨酸取代；
(vi)第15位的苏氨酸被丝氨酸取代。
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芋螺毒素肽κ-CPTx-btl01，其氨基酸序列为：KCCTMSVCQPPPVCTCCA(即SEQ ID NO：1)，其分子量为1872.72道尔顿。
发明人还发现，通过对SEQ ID NO：1所示氨基酸序列的一个或多个特定氨基酸残基进行特定的取代，还可以保留其原有的功能，即为所述(b)多肽。
第二方面，本发明提供了编码如第一方面所述的芋螺毒素肽κ-CPTx-btl01的多核苷酸。
在具体实施方案中，当芋螺毒素肽κ-CPTx-btl01具有如SEQ ID NO：1所示氨基酸序列时，编码其的多核苷酸具有如SEQ ID NO：2所示的核苷酸序列，即其核苷酸序列为：AAGTGCTGTACTATGTCGGTATGCCAGCCGCCGCCAGTGTGTACATGCTGCGCT。
第三方面，本发明提供了一种核酸构建体，其包含如第二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。
第四方面，本发明提供了一种表达载体，其包含如第三方面所述的核酸构建体。
第五方面，本发明提供了一种转化的细胞，其中转化了如第三方面所述的核酸构建体或如第四方面所述的表达载体。
第六方面，本发明提供了如第一方面所述的芋螺毒素肽κ-CPTx-btl01在制备抑制钙离子通道的药物中的应用；优选地，所述钙离子通道为高电压激活钙离子通道。
发明人研究了本发明芋螺毒素肽κ-CPTx-btl01的生物学活性。具体地，采用全细胞膜片钳方法检测本发明芋螺毒素肽κ-CPTx-btl01对背根神经节细胞(DRG细胞)离子通道的作用。实验中，给予两个去极化刺激，第一个从-90mV到-30mV，刺激250ms，第二个从-60mV到0mV，刺激250ms，两个刺激的间隔是500ms，同时记录LVA钙通道和HVA钙通道。通过与阳性对照物硝苯地平(Nifedipine，第一代钙通道阻滞剂，为抗高血压、防治心绞痛药物，是20世纪80年代中期世界畅销的药物之一)的活性比较分析，确定其钙离子通道抑制效果及其后续的应用方向。
发明人通过上述实验发现，本发明芋螺毒素肽κ-CPTx-btl01能够有效抑制高电压激活钙离子通道(HVA)电流，抑制效果比阳性对照硝苯地平更高。
第七方面，本发明提供了如第一方面所述的芋螺毒素肽κ-CPTx-btl01在制备治疗心血管系统疾病药物中的应用；优选地，所述心血管系统疾病为高血压、心绞痛或冠心病。
根据第六方面所述，由于本发明芋螺毒素肽κ-CPTx-btl01能够有效抑制高电压激活钙离子通道(HVA)电流，因而可应用于治疗高血压、心绞痛、冠心病等疾病。
第八方面，本发明提供了一种药物组合物，其包含如第一方面所述的芋螺毒素肽κ-CPTx-btl01及药学上可接受的载体。
第九方面，本发明提供了如第一方面所述的芋螺毒素肽κ-CPTx-btl01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，按其氨基酸序列合成芋螺毒素肽κ-CPTx-btl01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，当芋螺毒素肽κ-CPTx-btl01为如SEQ ID NO：1所示氨基酸序列的多肽时，还可以通过从天然生物体中提取获得。具体地，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所示氨基酸序列的芋螺毒素肽κ-CPTx-btl01：
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
本发明的芋螺毒素肽κ-CPTx-btl01可来源于天然活性动物资源，属于钙通道阻滞剂，与传统小分子药物相比，具有高选择性、高亲脂性和高安全性等优点；又因其结构简单、易于人工合成，能有效抑制钙通道电流且其活性比硝苯地平更高，因此可广泛用于钙离子通道相关疾病的治疗，例如，在临床上对于治疗高血压、冠心病、心绞痛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-btl01的质谱序列鉴定结果；
图2显示10μM芋螺毒素肽κ-CPTx-btl01对高电压激活钙通道的抑制作用；
图3显示10μM硝苯地平对高电压激活钙通道的抑制作用。
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  本发明芋螺毒素肽κ-CPTx-btl01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：buffer A：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12cm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-btl01的质谱序列搜索比对结果如图1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KCCTMSVCQPPPVCTCCA的κ-CPTx-btl01多肽。
实施例2  本发明芋螺毒素肽κ-CPTx-btl01的化学合成
应用芴甲氧羰基(Fmoc)固相化学合成的方法合成如SEQ ID NO：1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-btl01对钙离子通道的抑制活性
采用膜片钳技术，检测本发明芋螺毒素肽κ-CPTx-btl01对钙离子通道的抑制活性。具体地，称取实施例2中制备的、复性后的芋螺毒素肽κ-CPTx-btl01，通过全细胞膜片钳方法检测芋螺毒素肽κ-CPTx-btl01对大鼠背根神经节(DRG)细胞离子通道的作 用。以硝苯地平为阳性对照。
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，给予细胞两个去极化刺激，第一个从-90mV到-30mV，刺激250ms，第二个从-60mV到0mV，刺激250ms，两个刺激的间隔是500ms，同时记录LVA钙通道和HVA钙通道。向细胞外液中加入多肽样品使其有效浓度达10μM同时观察记录钙通道电流的变化(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。
芋螺毒素肽κ-CPTx-btl01的检测结果如图2所示，硝苯地平的检测结果如图3所示，10μM的κ-CPTx-btl01对DRG神经元细胞HVA钙通道电流的抑制率为52.17％(如表1)。上述结果显示：相同浓度的κ-CPTx-btl01与硝苯地平比较，对HVA钙通道电流的抑制率更高，提示其可以作为降压药的候选药物。
表1、芋螺毒素肽κ-CPTx-btl01对钙离子通道抑制率的膜片钳检测结果
Figure PCTCN2014095195-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 (10)

  1. 一种芋螺毒素肽κ-CPTx-bt101，其为：
    (a)如SEQ ID NO：1所示氨基酸序列的多肽；或者，
    (b)如SEQ ID NO：1所示氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的，具有(a)多肽功能的由(a)衍生的多肽；所述被取代、添加或缺失的氨基酸中不包括半胱氨酸；
    所述(a)和(b)多肽的氨基酸序列内含三对二硫键。
  2. 如权利要求1所述的芋螺毒素肽κ-CPTx-bt101，其特征在于，所述一个或多个氨基酸的取代选自由以下组成的组：
    (i)第1位的赖氨酸被精氨酸取代；
    (ii)第4位的苏氨酸被丝氨酸取代；
    (iii)第7位的缬氨酸被亮氨酸或异亮氨酸取代；
    (iv)第9位的谷氨酰胺被天冬酰胺取代；
    (v)第13位的缬氨酸被亮氨酸或异亮氨酸取代；
    (vi)第15位的苏氨酸被丝氨酸取代。
  3. 编码如权利要求1或2所述芋螺毒素肽κ-CPTx-bt101的多核苷酸。
  4. 一种核酸构建体，其包含权利要求3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。
  5. 一种表达载体，其包含权利要求4所述的核酸构建体。
  6. 一种转化的细胞，其中转化了权利要求4所述的核酸构建体或权利要求5所述的表达载体。
  7. 如权利要求1或2所述的芋螺毒素肽κ-CPTx-bt101在制备抑制钙离子通道的药物中的应用；优选地，所述钙离子通道为高电压激活钙离子通道。
  8. 如权利要求1或2所述的芋螺毒素肽κ-CPTx-bt101在制备治疗心血管系统疾病 药物中的应用；优选地，所述心血管系统疾病为高血压、心绞痛或冠心病。
  9. 一种药物组合物，其包含权利要求1或2所述的芋螺毒素肽κ-CPTx-bt101及药学上可接受的载体。
  10. 如权利要求1或2所述的芋螺毒素肽κ-CPTx-bt101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，按其氨基酸序列合成芋螺毒素肽κ-CPTx-bt101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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