WO2018086540A1 - 森林山蛭抗血栓肽Sylvestin及其应用 - Google Patents

森林山蛭抗血栓肽Sylvestin及其应用 Download PDF

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WO2018086540A1
WO2018086540A1 PCT/CN2017/109962 CN2017109962W WO2018086540A1 WO 2018086540 A1 WO2018086540 A1 WO 2018086540A1 CN 2017109962 W CN2017109962 W CN 2017109962W WO 2018086540 A1 WO2018086540 A1 WO 2018086540A1
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amino acid
acid sequence
hawthorn
sylvestin
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赖仞
刘伟慧
龙承波
吕秋敏
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中国科学院昆明动物研究所
昆明龙津药业股份有限公司
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Priority to US16/347,954 priority Critical patent/US20190284258A1/en
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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
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6451—Coagulation factor XIIa (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21038—Coagulation factor XIIa (3.4.21.38)

Definitions

  • the invention provides a forest hawthorn (Haemadipsa sylvestris) antithrombotic peptide Sylvestin and a variant thereof, and an application thereof, and belongs to the field of biomedical technology.
  • Cerebrovascular disease is one of the main causes of death in middle-aged and elderly people in China, and it is also one of the research priorities of the world's health strategy.
  • cerebrovascular diseases the incidence of acute ischemic diseases ranks first, with high morbidity, high mortality and high recurrence rate.
  • the global stroke has more than 1.5 million deaths per year, and it is increasing year by year with the increase of the elderly population, which seriously threatens human life and quality of life. Therefore, research on drugs for the treatment of acute cerebral ischemia has enormous social needs and important social significance.
  • the main cause of acute cerebral ischemia is cerebral vascular embolism, which is the result of vascular infarction caused by thrombosis in the cerebral blood vessels leading to poor blood flow.
  • cerebral vascular embolism is the result of vascular infarction caused by thrombosis in the cerebral blood vessels leading to poor blood flow.
  • One of the key measures for the treatment of acute cerebral ischemia is to relieve vascular embolism and restore blood supply to the ischemic area. Therefore, thrombolytic therapy has become a key link in the clinical treatment of acute cerebral ischemia.
  • the application of thrombolytic drugs has been clinically proven to effectively relieve cerebral ischemia and become the main direction for the treatment of acute cerebral ischemia.
  • the anti-cerebral ischemic drug approved by the US FDA has only one type of t-PA, but the drug is only effective for 3 hours.
  • t-PA The target of t-PA is plasminogen, which leads to a higher incidence of bleeding.
  • Drugs with FXIIa and kallikrein as targets have a very low bleeding tendency, so they are used as targets to study and develop new drugs with low efficacy and low side effects.
  • Anti-thrombotic drugs have good social and economic benefits and market prospects.
  • the mites are a traditional Chinese medicine in China, which was described in the Shennong Bencao Classic more than 2,000 years ago. "Compendium of Materia Medica” says: “Salty blood, bitterness wins blood. The salty bitterness of the water scorpion, in addition to blood storage, is the medicine of the liver and blood, so it can pass the liver and collect blood.” Chinese medicine believes that leeches is a traditional break. Blood medicine, which has the effects of sputum, sputum, and waterway, is mainly used for the treatment of blood stasis, stroke, hemiplegia, bruises and other diseases. In 2005, Europe officially approved steroid therapy as a legal treatment. There are only 350,000 leeches in Germany for medical treatment each year.
  • Forest foothills also known as grasshoppers. ⁇ , ⁇ .
  • the body is slightly elliptical and is about 3 cm long.
  • the model's model is produced in Changlin Mountain, Sri. Distributed in Indonesia, Sri, India, Vietnam and mainland China and other places in Yunnan. Mainly inhabit wet mountain meadows or bamboo forests and near water or water. When the human and animal pass, they attach to the blood. Hawthorn can secrete anticoagulant substances and destroy blood clotting function. Therefore, the wounds bitten by the hawthorn often bleed. The folks also use this property to treat the patient's local blood flow with hawthorn.
  • the forest hawthorn antithrombotic peptide Sylvestin according to the present invention is capable of inhibiting thrombus formation. It was confirmed by animal models that it has strong antithrombotic activity and inhibits acute cerebral ischemic activity.
  • the inventors searched and compared the full-sequence amino acids of the anti-thrombotic peptide Sylvestin of the present invention through a protein database, and found no identical polypeptide.
  • the inventors searched and compared the coding sequences of the forest hawthorn antithrombotic peptide Sylvestin of the present invention through a gene database, and found no identical genes.
  • the object of the present invention is to provide a novel polypeptide having strong antithrombotic properties, Sylvestin, and variants thereof, as well as its prophylactic and therapeutic applications, based on the above-mentioned unexpected findings by the inventors.
  • the present invention provides the following technical solutions:
  • the molecular weight of the antithrombotic peptide Sylvestin is 4790.5 Daltons, and its amino acid sequence (EQ ID NO: 1) is: TSEPVCACPK MLFWVCGKDG ETYTHPCIAK CHNVEVEHDG KCK.
  • Cloning of the anti-thrombotic peptide corresponding cDNA includes:
  • RNA extraction, mRNA purification, mRNA reverse transcription and cDNA library construction of forest hawthorn, primers were designed, and the anti-thrombotic peptide gene of forest hawthorn was screened by PCR.
  • the amplification primer was 20 nucleotides in length, the sequence was 5' AAACCTCGGAACCGGTATGT 3', and the other amplification primer of PCR was a 3' PCR primer, and the sequence was 5'CCGAGGTTTGGTGGCTCATT 3'.
  • the positive monoclonal obtained was subjected to nucleotide sequence determination.
  • the sequencing results indicated that the DNA encoding the forest hawthorn antithrombotic peptide consisted of 316 nucleotides, and the sequence from the 5' end to the 3' end (nucleotide sequence SEQ ID NO: 2) was:
  • the nucleotide fragment of 55-183 encodes a mature peptide of the forest hawthorn antithrombotic peptide.
  • the collected forest hawthorn homogenate was first passed through a gel chromatography column Sephadex G-50, and the peak with antithrombotic activity was collected, lyophilized, and subjected to reverse high pressure liquid phase (RP-HPLC) C4 and C18 columns to obtain a forest.
  • RP-HPLC reverse high pressure liquid phase
  • the sequence and the amino acid sequence deduced from the forest hawthorn antithrombotic peptide gene were determined according to the Edman degradation method, and the entire sequence was synthesized using an automatic peptide synthesizer (433A, Applied Biosystems). It was desalted by HPLC reverse column chromatography to confirm its purity was greater than 95%. The molecular weight was determined by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). The synthetic antithrombotic peptide is dissolved in sterile water for activity assays.
  • MALDI-TOF matrix-assisted laser desorption ionization time of flight mass spectrometry
  • the forest hawthorn anti-thrombotic peptide Sylvestin was isolated and purified, and its cDNA sequence was cloned.
  • the antithrombotic peptide can inhibit the function of FXIIa and kallikrein, has extremely significant antithrombotic effect and inhibits acute cerebral ischemia.
  • the antithrombotic peptide has the advantages of simple structure, convenient artificial synthesis and strong antithrombotic activity, and can be applied to preparing an agent for inhibiting FXIIa and kallikrein, and preparing an antithrombotic and treating acute cerebral ischemia.
  • Figure 1 shows the antithrombotic effect of the forest hawthorn antithrombotic peptide Sylvestin.
  • Figure 2 shows the anti-acute cerebral ischemia effect of the forest hawthorn antithrombotic peptide Sylvestin.
  • the first step was Sephadex G-50 gel chromatography. 2 ml of hawthorn homogenate was taken and applied to a Sephadex G-50 (Amersham Bioscience) gel column (26 mm x 100 cm) equilibrated with Tris-HCl (0.02 mol/L, pH 7.8) buffer. Elution was carried out with the same concentration of equilibration buffer at a flow rate of 0.3 ml/min, 3 ml/tube, and collected using a CBS-A programmable fully automatic partial collector (Shanghai Qingpu Huxi Instrument Factory). The 280 nm and 215 nm values were determined using an Ultrospec 2100pro spectrophotometer (Amersham Biosciences). The peak fractions were collected and stored at -20 ° C until use.
  • the second step C4 reverse phase high pressure chromatography.
  • the active protein was further separated using a reversed phase high pressure chromatography (Waters 1525 Binary HPLC Pump) C4 column (Lichrospher 10 x 250 mm); solvent A: ultrapure aqueous solution of 0.1% TFA, solvent B: 0.1% TFA in acetonitrile. Elution uses a linear concentration gradient: 0-10 min, B: 0%; 10-11 min, B: 0-5%; 11-40 min, B: 5-33%; 40-50 min, B: 33-38%; -60 min, B: 38-70%; 60-70 min, B: 70-100%.
  • the flow rate was 1.5 ml/min.
  • the loading amount was 3 mg of crude protein. Peak collection was detected using a Waters 2489 Visible/UV detector (215 nm) with each peak being one collection unit.
  • the third step C8 column reversed-phase high pressure chromatography.
  • C8 column (X Bridge TM 4.6 ⁇ 250mm) .
  • Solvent A Ultrapure aqueous solution of 0.1% TFA
  • solvent B 0.1% TFA in acetonitrile. Elution uses a linear concentration gradient: 0-10 min, B: 0%; 10-14 min, B: 0-20%; 14-24 min, B: 20%; 24-55 min, B: 20-35%; 55-60 min , B: 35-100%.
  • the flow rate was 0.7 ml/min and the loading amount was 1 mg of crude protein. Peak collections were detected using a Waters 2489 Visible/UV detector (215 nm) with each peak being one collection unit.
  • the above steps track the detection and collection of FXIIa and kallikrein inhibitory active fractions.
  • the purified forest hawthorn antithrombotic peptide obtained by the Edman degradation method was subjected to N-terminal sequencing (model 491, ABI, USA).
  • the molecular weight of the antithrombotic peptide was determined by electrospray ionization mass spectrometry (ESI-MS).
  • PCR amplification was performed as follows:
  • the synthesized cDNA double strands were dispensed into 10 ⁇ L of each tube by a PCR tube, and 5 ⁇ L was taken out for 1% agarose electrophoresis, and the others were immediately stored at -80 ° C.
  • Each cell pellet was resuspended in 600 ⁇ L of pre-cooled 0.1 M CaCl2-MgCl 2 solution (80 mM MgCl 2 , 20 mM CaCl 2 ) per 1 mL of the initial culture.
  • the amplification primer was 20 nucleotides in length, the sequence was 5' AAACCTCGGAACCGGTATGT 3', and the other amplification primer was 3' primer, and the sequence was 5'CCGAGGTTTGGTGGCTCATT 3'.
  • sequencing was performed using an ABI 3730 sequencer.
  • the sequencing primer was M13(+): 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', M13(-): 5'-GAGCGGATAACAATTTCACACAGG-3'. The sequencing results were sequenced.
  • nucleotide sequence SEQ ID NO: 2 The cDNA sequencing results from the 5' end to the 3' end sequence (nucleotide sequence SEQ ID NO: 2) are:
  • the nucleotide sequence of the cDNA encoding the forest hawthorn antithrombotic peptide Sylvestin cloned in this example has the following characteristics: the sequence length is 316 bases, the sequence type: nucleic acid, number of strands: single strand, topology : linear, sequence type: cDNA, source: forest haw.
  • the coding region of the mature antithrombotic peptide Sylvestin of the forest hawthorn was nucleotides 55-183 of the nucleotide sequence SEQ ID NO: 2.
  • the amino acid sequence (SEQ ID NO: 1) of the forest hawthorn antithrombotic peptide Sylvestin is:
  • the polynucleotide encoding the antithrombotic peptide Sylvestin is used as a genetic engineering preparation of an antithrombotic peptide.
  • the antithrombotic peptide Sylvestin is used in the preparation of FXIIa and kallikrein inhibitors and anti-thrombotic and anti-acute cerebral hemorrhage drugs.
  • Cs+ acts as a bombardment particle with a current of 1 ⁇ A and an emission voltage of 25 KV.
  • Purified forest hawthorn antithrombotic peptide was identified by high performance liquid chromatography-HPLC method. The molecular weight was determined by fast atom bombardment mass spectrometry, isoelectric focusing electrophoresis to determine the isoelectric point, and the amino acid sequence structure was determined by automatic amino acid sequencer.
  • the forest hawthorn antithrombotic peptide Sylvestin is a polypeptide encoded by the forest hawthorn antithrombotic peptide gene with a molecular weight of 4790.5 Daltons and an isoelectric point of 6.28.
  • the amino acid sequence (SEQ ID NO: 1) of the antithrombotic peptide Sylvestin is: TSEPVCACPK MLFWVCGKDG ETYTHPCIAK CHNVEVEHDG KCK.
  • 10 ⁇ L of the sample in a 96-well plate was mixed with 10 ⁇ L of a final concentration of 0.2 ⁇ M of FXIIa in 40 ⁇ L of a buffer (100 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM CaCl 2 ). After standing at room temperature for 5 minutes, a mixture of 30 ⁇ L of buffer and 10 ⁇ L of a final concentration of 0.04 mM chromogenic substrate was added in a final volume of 100 ⁇ L. The kinetics of the coagulation reaction was measured using an Epoch (BioTek) plate reader, GEN CHS 1.09 software for OD 405 nm, 20 min, at intervals of 47 s.
  • the sample concentration was 10 ⁇ M.
  • the Sylvestin synthesized in Example 3 had no inhibitory effect on thrombin, plasminogen FXa and the like. Can inhibit FXIIa and kallikrein.
  • the inhibition constants of Sylvestin on FXIIa and kallikrein were calculated to be 2.9 ⁇ M and 17.8 nM, respectively.
  • the APTT reagent was equilibrated to room temperature and the APTT reagent was mixed by gentle inversion. After mixing 50 ⁇ L of PLT reagent, 50 ⁇ L of normal plasma and 5 ⁇ L of the sample, incubate for 3 minutes in a 37 ° C water bath, add 50 ⁇ L of preheated CaCl 2 solution, mix immediately, and measure OD650nm with a microplate reader.
  • PT experiment Pre-warm prothrombin reagent for 15 minutes at 37 °C.
  • carrageenan carrageenan, type I, Sigma, dissolved in physiological saline to 1% concentration
  • the thrombus formation rate was >90 due to the low temperature environment. %, so the feeding temperature is 17.5 °C.
  • the average length of thrombus formation was determined according to the color change of the tail skin. Referring to Figure 1, the average length of thrombus in each group increased with time. 12h due to inconspicuous thrombosis, there was a large error in the measurement, but the control group was significantly more obvious than the other groups; heparin sodium doubled from 12h to 24h; the control group remained basically the same, probably because it was limited to the tail Length; sample group length increased slightly.
  • Kunming mice (30-35g) were anesthetized with 2% sodium pentobarbital (80mg/kg). After deep anesthesia, the cervical median incision was made to cut the skin and subcutaneous tissue of the rat layer, and the sternocleidomastoid muscle was isolated and cut off. The anterior abdominal abdomen was exposed, and the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were exposed. The thyroid artery and pharyngeal artery on the ECA were coagulated by an electric coagulator and cut.
  • CCA common carotid artery
  • ICA internal carotid artery
  • ECA external carotid artery
  • Cerebral ischemia for 24h according to the Bederson Score score, and the neurological deficit score was evaluated and scored: 0 was divided into no dysfunction; 1 was unable to extend the right forelimb; 2 was rotated to the right; 3 was divided to the right; 4 points For lack of autonomic activity with disturbance of consciousness; 5 is divided into death.
  • the head was removed by anesthesia, the brain tissue was taken out, placed in a brain mold (on ice), and cut into 2 mm thick sections along the optic chiasm, and placed in 2% TTC phosphate buffer at 37 ° C. The cells were stained for 30 min in a thermostat and fixed overnight with 4% paraformaldehyde and photographed.
  • ischemic volume [ischemic volume - (left hemi-brain volume - right hemi-brain volume)] / right hemi-brain volume x 100.
  • the tail vein was administered once, the experimental group was 1, 3, 5 mg/kg Sylvestin (synthesized according to Example 2), and the control group was physiological saline in a volume of 100 ⁇ l.
  • the tail vein was administered once, at the same dose as above; once again at 6 h. 6 in each group. It was found that Sylvestin can effectively alleviate the damage caused by cerebral ischemia and reperfusion (Fig. 2).

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Abstract

本发明提供了一种森林山蛭（Haemadipsa sylvestris）抗血栓肽Sylvestin及其应用，属于生物医学技术领域。所述森林山蛭抗血栓肽Sylvestin能够抑制FXIIa和kallikrein，具有抗血栓/梗塞作用并减轻脑缺血造成的损伤，可应用于制备FXIIa、kallikrein的抑制剂及抗血栓/梗塞、抗脑缺血损伤的药物。

Description

森林山蛭抗血栓肽Sylvestin及其应用
本申请要求于2016年11月08日提交中国专利局、申请号为201610979895.7、发明名称为“森林山蛭抗血栓肽Sylvestin及其基因和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明提供一种森林山蛭(Haemadipsa sylvestris)抗血栓肽Sylvestin及其变体以及其应用，属于生物医学技术领域。
背景技术
脑血管疾病是导致我国中老年人死亡的主要病因之一，也是世界性卫生战略研究重点之一。在脑血管疾病中，急性缺血性疾病的发病率居于首位，具有致残率高、死亡率高和复发率高的特点。全球脑卒中病死逾150万例/年，随着老年人口的增加呈逐年增长的趋势，严重威胁着人类生命健康和生活质量。因此，研究治疗急性脑缺血的药物具有巨大的社会需求和重要的社会意义。
急性脑缺血的主要病因是脑血管栓塞，这是由于脑血管中血栓形成导致血流不畅，进而造成血管梗死的结果。治疗急性脑缺血的关键措施之一是解除血管栓塞，恢复缺血区血液供应。因此，溶栓治疗成为临床治疗急性脑缺血的关键环节。溶栓药物的应用被临床证明能有效解除脑缺血，成为治疗急性脑缺血的主要方向。目前被美国FDA批准的抗脑缺血药物只有t-PA一种，但该药仅在发病3h有效。t-PA的作用靶点是纤溶酶原，会导致较高的出血发生率。以FXIIa和kallikrein为作用靶点的药物出血倾向非常低，因此以它们为作用靶点，研究和开发新型药效好、副作用低的治 疗抗血栓药物，具有良好的社会经济效益和市场前景。
蛭类是我国的一种传统中药，两千多年前的《神农本草经》中就有描述。《本草纲目》谓其：“咸走血，苦胜血。水蛭之咸苦，以除蓄血，乃肝经血分药，故能通肝经聚血。”中医认为水蛭是一种传统的破血药，有逐瘀、通经脉、利水道的功效，主要用于治疗血瘀经闭、中风偏瘫、跌打损伤等疾病。2005年，欧洲正式批准蛭类疗法为合法的治疗手段。每年仅在德国就有35万条水蛭用于医疗。
森林山蛭(Haemadipsa sylvestris)，又称草蛭。蛭纲,水蛭科。体略呈长椭圆形,长约3厘米。该物种的模式产地在缅甸长林山。分布于印度尼西亚、缅甸、印度、越南以及中国大陆的云南等地。主要栖息于潮湿的山区草地或竹林里以及水附近或水中。当人畜经过时，就附着吸食血液。山蛭能分泌抗凝血的物质，破坏凝血功能。因此被山蛭咬过的伤口常流血不止。民间也利用这一性质，用山蛭来治疗病人的局部血流不畅。
我们首次从森林山蛭中发现了一种作用很强的FXIIa和kallikrein抑制剂，其具有抗血栓/抗急性脑出血功能，命名为Sylvestin(以下称森林山蛭抗血栓肽)。
本发明涉及的森林山蛭抗血栓肽Sylvestin能够抑制血栓形成。通过动物模型证实它具有很强的抗血栓和抑制急性脑缺血活性。发明人将本发明的抗血栓肽Sylvestin全序列氨基酸经蛋白质数据库进行搜寻比较，未发现有任何相同多肽。发明人将本发明的森林山蛭抗血栓肽Sylvestin的编码序列经基因数据库进行搜寻比较，未发现有任何相同基因。
发明内容
本发明的目的是基于发明人的上述意外发现，提供一种新的具有强烈的抗血栓性的多肽Sylvestin及其变体，以及其预防和治疗性应用。
为了实现本发明的目的，本发明提供了如下技术方案：
抗血栓肽Sylvestin的分子量为4790.5道尔顿，其氨基酸序列(EQ ID NO:1)为：TSEPVCACPK MLFWVCGKDG ETYTHPCIAK CHNVEVEHDG KCK。
抗血栓肽对应cDNA的克隆包括:
森林山蛭总RNA提取，mRNA纯化，mRNA反转录及cDNA文库构建，设计引物，利用PCR方法筛选森林山蛭抗血栓肽基因。扩增引物长度为20个核苷酸，其序列为5’AAACCTCGGAACCGGTATGT 3’，PCR另一扩增引物为3’PCR引物，其序列为5’CCGAGGTTTGGTGGCTCATT 3’。所获阳性单克隆进行核苷酸序列测定。测序结果表明编码森林山蛭抗血栓肽的DNA由316个核苷酸组成，自5’端至3’端序列(核苷酸序列SEQ ID NO:2)为:
Figure PCTCN2017109962-appb-000001
其中55-183的核苷酸片段编码森林山蛭抗血栓肽的成熟肽。
森林山蛭抗血栓肽的分离纯化方法：
收集的森林山蛭匀浆液，首先过凝胶层析柱Sephadex G-50，收集具有抗血栓活性的峰，冻干，过反向高压液相(RP-HPLC)C4、C18柱，纯化得到森林山蛭抗血栓肽。
森林山蛭抗血栓肽的化学合成方法：
根据Edman降解法测得序列和编码森林山蛭抗血栓肽基因推断的氨基酸序列，用自动多肽合成仪(433A,Applied Biosystems)合成其全序列。通过HPLC反向柱层析脱盐纯化，并确定其纯度大于95％。用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)测定其分子量。合成的抗血栓肽溶于灭菌水，用于活性测定。
本发明的有益效果在于:
分离纯化得到森林山蛭抗血栓肽Sylvestin，并克隆得到其cDNA序列。该抗血栓肽能够抑制FXIIa、kallikrein的功能，有极显著的抗血栓和抑制急性脑缺血作用。另外该抗血栓肽具有结构简单、人工合成方便、抗血栓活性强的优点，能应用于制备抑制FXIIa、kallikrein的试剂和制备抗血栓、治疗急性脑缺血的药物。
附图说明
图1显示森林山蛭抗血栓肽Sylvestin的抗血栓作用。
图2显示森林山蛭抗血栓肽Sylvestin的抗急性脑缺血作用。
具体实施方式
下面用实施例来进一步说明本发明的实质性内容，但本发明的内容并不局限于此。
实施例1 森林山蛭抗血栓肽Sylvestin的分离纯化
1.1山蛭的处理
用液氮直接速冻活的森林山蛭。在低温下，用棉布包裹山蛭并用铁 锤把山蛭敲成数段。把敲过的山蛭和适量的50mM Tris-HCl(pH 8.0)缓冲溶液混合匀浆，4℃，12000g离心30min，上清即为森林山蛭体腔液粗样。把全部上清混合在一起，分装成3ml每管，于-80℃保存。
1.2Sephadex G-50凝胶分离与反相高压色谱分离
第一步，Sephadex G-50凝胶层析。取2ml山蛭匀浆液，上样于Tris-HCl(0.02mol/L，pH 7.8)缓冲液平衡过的Sephadex G-50(Amersham Bioscience)凝胶柱(26mm×100cm)。用同样浓度的平衡缓冲液洗脱，流速为0.3ml/min，3ml/管，使用CBS-A程控全自动部分收集器收集(上海青浦沪西仪器厂)。使用Ultrospec 2100pro分光光度计(Amersham Biosciences)测定280nm和215nm值。收集各峰组分存于-20℃备用。
第二步，C4反相高压色谱法。活性蛋白使用反相高压色谱(Waters1525 Binary HPLC Pump)C4柱(Lichrospher 10×250mm)继续分离；溶剂A：0.1％TFA的超纯水溶液，溶剂B：0.1％TFA的乙腈溶液。洗脱使用线性浓度梯度：0-10min，B：0％；10-11min，B：0-5％；11-40min，B：5-33％；40-50min，B：33-38％；50-60min，B：38-70％；60-70min，B：70-100％。流速为1.5ml/min。上样量为3mg粗样蛋白。峰收集使用Waters2489可见/紫外检测器检测(215nm)，每一个峰为一个收集单位。
第三步，C8柱反相高压色谱法。C8柱(X BridgeTM 4.6×250mm)。溶剂A：0.1％TFA的超纯水溶液，溶剂B：0.1％TFA的乙腈溶液。洗脱使用线性浓度梯度：0-10min，B：0％；10-14min，B：0-20％；14-24min，B：20％；24-55min，B：20-35％；55-60min，B：35-100％。流速为0.7ml/min，上样量为1mg粗样蛋白。峰收集使用Waters 2489可见/紫外检测器检测(215nm)，每一个峰为一个收集单位。以上步骤跟踪检测和收集FXIIa和kallikrein抑制活性部分。Edman降解法对其纯化所得的森林山蛭抗血栓肽纯品进行 N-端测序(model 491，ABI，美国)。电喷雾电离质谱(ESI-MS)测定抗血栓肽分子量。
实施例2 森林山蛭抗血栓肽cDNA克隆
2.1森林山蛭总RNA提取
(1)迅速从液氮中取出多条山蛭，用剪刀剪下山蛭前端(包括山蛭口腔和咽部)，总重量100mg左右，放入研钵中，加入液氮后充分研磨。待研成粉末状后，加入1ml Trizol提取缓冲液(Invitrogen)，与液氮共研磨。待Trizol溶化，将研钵中全部液体吸出至1.5ml离心管中，室温放置5min。
(2)加入200μL氯仿，剧烈震荡混匀15s，室温放置5min；4℃，12000g离心10min；吸取上层无色水相至新的1.5ml离心管中(尽量多吸上层液体，但一定要避免吸到中下层)。
(3)加入等体积4℃预冷异丙醇，颠倒混匀，室温放置15min，4℃，12000g离心10min，管底微量沉淀为RNA，用移液枪小心吸去上清。
(4)沉淀加入1ml 75％冰上预冷乙醇洗涤，4℃，7500g离心5min，弃上清，重复两次；超净台中放置5-10min，晾干(无乙醇味)，即为森林山蛭总RNA；往晾干的EP管中加入30μL 0.1％DEPC水，轻微震荡溶解RNA。从中吸取2μL加98μL DEPC水，于230nm、260nm、280nm波长处检其吸光值。取10μL用于1％琼脂糖胶电泳。其余总RNA于-80℃冻存。
2.2cDNA文库的构建：
按照CreatorTM SMARTTM cDNA Library Construction Kit(Clontech)说明书操作构建森林山蛭头部cDNA文库。具体操作如下：
(1)cDNA第一链合成(mRNA反转录)
在无RNase PCR管中加入2μL森林山蛭总RNA、1μL SMARTTMⅣoligonucleotide、1μL CDSⅢ/3’Primer，加1μL DEPC水使总体积达到5μL，混匀并离心10s；72℃保温2min，冰上孵育2min；在上述PCR管中加入2μL 5×第一链buffer、1μL 20mM DTT、1μL 10mM dNTP混合物、1μL PowerScript逆转录酶，混匀并离心10s。PCR仪中42℃保温1h，冰上终止反应。
(2)采用长末端聚合酶链式反应(LD-PCR)方法扩增cDNA第二链
将1μL cDNA第一链(mRNA反转录)，40μL去离子水，5μL 10×buffer缓冲，1μL 50×dNTP混合物，1μL 5’PCR primer，1μL CDS Ⅲ/3’PCR引物以及1μL聚合酶于PCR管中混匀。按以下程序进行PCR扩增：
①95℃ 1min
②20个循环：
95℃ 15sec，
65℃ 30sec，
68℃ 6min。
扩增结束后，合成好的cDNA双链用PCR管分装成10μL每管，取出5μL进行1％琼脂糖电泳，其余立刻置于-80℃保存。
2.3大肠杆菌DH5α感受态细胞的制备：
(1)挑取单个DH5α菌落，接种于1mL不含氨苄青霉素的LB液体培养基中，37℃培养过夜，次日取上述菌液按比例1:100再接种于1mL LB培养液中，37℃振荡2h。
(2)当OD600达到0.35时，将菌液在冰上放置10min使培养物冷却至0℃。
(3)4℃，5000rpm离心5min，以回收细胞。
(4)倒出培养液，将管倒置lmin以使最后的痕量培养液流尽。
(5)每1mL初始培养液加入600μL预冷的0.1M CaCl2-MgCl2溶液(80mM MgCl2,20mM CaCl2)重悬每份细胞沉淀。
(6)4℃，5000rpm离心5min，以回收细胞。
(7)倒出培养液，将管倒置lmin以使最后的痕量液体流尽。
(8)每1mL初始培养物加入60μL用冰预冷的0.1M CaCl2重悬细胞沉淀。4℃冰箱放置10-18h。
2.4cDNA文库的筛选：
(1)特异性引物的合成
使用primer blast设计两条引物，委托生工公司合成。扩增引物长度为20个核苷酸，其序列为5’AAACCTCGGAACCGGTATGT 3’，另一扩增引物为3’引物，其序列为5’CCGAGGTTTGGTGGCTCATT 3’。
(2)从山蛭cDNA文库中克隆目标序列
20μL体系：Taq酶0.1μL，CDSIII、SMART4各0.4μL，dNTP 0.4μL，buffer 2μL，Mg2+1.2μL，PCR水16μL
PCR条件：
Figure PCTCN2017109962-appb-000002
(3)序列的连接、转化与检测。在微量离心管中加入0.2μL Takara  PMD19-T载体，2.3μL DNA双链；加入2.5μL等量的连接酶缓冲液混合物(冰上溶解)；16℃反应过夜；全部5μL连接产物加至60μLDH5α感受态细胞中，冰上放置30min；42℃热激90s，轻放于冰上3-5min,修复细胞膜；尽快加入温浴过的LB培养基，37℃，80rpm摇菌45min；取100μL涂布与含有100ug/ml的AMP LB培养基中，37℃培养16h；菌落长出后，挑单克隆进行10μL菌液PCR。
2.5挑取单克隆测序及序列筛选：
挑取20个与目标序列核酸大小相近的单菌落送测序公司进行DNA测序。采用ABI 3730测序仪测序。测序引物为M13(+)：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’,            M13(-):5’-GAGCGGATAACAATTTCACACAGG-3’。测序结果进行序列筛选。
2.6森林山蛭抗血栓肽Sylvestin DNA序列测定：
提取质粒DNA用双脱氧法测定核苷酸序列，使用仪器为美国Applied Biosystems373A全自动核苷酸序列测定仪，测序引物为BcaBESTTM Sequencing Primer RV-M和BcaBESTTM Sequencing Primer M13-47，BcaBESTTM Sequencing Primer RV-M序列：5`GAGCGGATAACAATTTCACACAGG 3’，BcaBESTTM Sequencing Primer M13-47：5’CGCCAGGGTTTTCCCAGTCACGAC 3’。cDNA测序结果自5’端至3’端序列(核苷酸序列SEQ ID NO:2)为：
Figure PCTCN2017109962-appb-000003
Figure PCTCN2017109962-appb-000004
如上所示，本实施例克隆出的编码森林山蛭抗血栓肽Sylvestin的cDNA的核苷酸序列有如下特征：序列长度为316个碱基，序列类型：核酸，链数：单链，拓扑学：直链状，序列种类：cDNA，来源：森林山蛭。
结合Edman降解法N端测序和质谱鉴定的结果进行分析，森林山蛭成熟抗血栓肽Sylvestin的编码区为核苷酸序列SEQ ID NO:2的第55-183位核苷酸。森林山蛭抗血栓肽Sylvestin的氨基酸序列(SEQ ID NO:1)为：
Figure PCTCN2017109962-appb-000005
编码抗血栓肽Sylvestin的多核苷酸作为基因工程制备抗血栓肽的应用。
抗血栓肽Sylvestin在制备FXIIa和kallikrein抑制剂和抗血栓、抗急性脑出血药物中的应用。
实施例3 森林山蛭抗血栓肽Sylvestin的化学合成
3.1抗血栓肽Sylvestin的化学合成方法：参见实施例2，根据编码森林山蛭抗血栓肽Sylvestin的蛋白测序结果和cDNA序列推断其氨基酸序列，用自动多肽合成仪合成其全序列。通过HPLC反相C18柱层析脱盐、纯化。
3.2分子量测定采用快原子轰击质谱法(Fast atom bombardment mass spectrometry,FAB-MS)，以甘油:间硝基苄醇:二甲亚砜(1:1:l，V:V:V，体积比)为底物，Cs+作为轰击粒子，电流为1μA，发射电压为25Kv。
3.3纯化的森林山蛭抗血栓肽用高效液相色谱HPLC方法鉴定其纯度，分子量测定采用快原子轰击质谱法，等电聚焦电泳测定等电点，用自动氨基酸测序仪测定氨基酸序列结构。
森林山蛭抗血栓肽Sylvestin是森林山蛭抗血栓肽基因编码的一种多肽，分子量4790.5道尔顿，等电点6.28。抗血栓肽Sylvestin的氨基酸序列(SEQ ID NO:1)为：TSEPVCACPK MLFWVCGKDG ETYTHPCIAK CHNVEVEHDG KCK。
实施例4 森林山蛭抗血栓肽Sylvestin的药理实验
4.1酶动力学：
在96孔板中10μL样品与10μL终浓度为0.2μM的FXIIa混合于40μL缓冲液(100mM Nacl、50mM Tris-HCl(pH8.0)、5mM CaCl2)中。室温放置5分钟后，加入30μL缓冲液与10μL终浓度为0.04mM发色底物的混合液，终体积为100μL。凝血反应的动力学使用Epoch(BioTek)酶标仪、GEN CHS 1.09软件检测OD405nm，20min，间隔47s。样品浓度为10μM。结果发现，实施例3合成的Sylvestin对凝血酶、纤溶酶原FXa等无抑制作用。可以抑制FXIIa和kallikrein。经计算Sylvestin对FXIIa和kallikrein的抑制常数分别为2.9μM、17.8nM。
4.2APTT和PT实验：
将APTT试剂平衡至室温，轻轻倒置混匀APTT试剂。50μLAPTT试剂、50μL正常血浆和5μL样品混匀后，在37℃的水浴锅中孵育3分钟，加入50μL预热的CaCl2溶液，立即混匀，用酶标仪记检测OD650nm。PT实验：37℃预热凝血酶原试剂15分钟。50μL正常血浆与5μL样品在37℃ 水浴锅中孵育3分钟，加入预热的凝血酶原试剂100μL，立即混匀，用酶标仪记录OD650nm。结果发现，实施例3合成的Sylvestin对PT实验无作用，对APTT实验具有浓度依赖效果，表明Sylvestin抑制内源性凝血途径，与作为FXIIa抑制剂的结果一致。
4.3卡拉胶致鼠尾血栓模型：
实验动物用昆明小鼠，体重25-30g(昆明医学院实验动物中心提供)，饲养一周后，随机分组(n＝6)，雌雄各半。第1组为生理盐水对照组，样品组为2mg/kg、4mg/kg实施例2合成的Sylvestin，阳性对照为500U/只肝素钠(北京鼎国昌盛生物技术有限责任公司)。尾静脉给药处理30min后，以60mg/kg的剂量从小鼠腹腔注射卡拉胶(carrageenan，type I，Sigma，用生理盐水溶解成1％的浓度)，由于在低温环境下，血栓形成率>90％，所以饲养温度为17.5℃。12、24、36、48h后根据尾部皮肤颜色变化测定血栓形成的平均长度。参见附图1，随着时间的增加，各组血栓的平均长度都增加了。12h由于血栓形成的不明显，测量时有较大的误差，但对照组明显比其他组症状明显；肝素钠从12h到24h长度增加了一倍；对照组基本不变，可能因为限于鼠尾的长度；样品组长度略微增加。在每个测量时间点，相对于对照组，Sylvestin和肝素对鼠尾血栓形成的影响都有统计学意义。数据处理为Graphpad Prime 5，数据平均值±SD，t-test(*p<0.05)。整体上随着时间的延长，Sylvestin对血栓形成的抑制率降低了，但即使48h后，Sylvestin处理组与对照组相比，仍有显著抑制作用。相对于肝素组和2mg/kg组，4mg/kg组抑制率变化不大。并且，Sylvestin对血栓的抑制率随着浓度的增加变大(图1)。
4.4急性脑缺血线栓模型：
使用2％戊巴比妥钠(80mg/kg)麻醉昆明小鼠(30～35g)，中深度麻醉后，颈正中切口逐层切开大鼠皮肤及皮下组织，分离胸锁乳突肌，切断二腹肌前腹，暴露右侧颈总动脉(CCA)，颈内动脉(ICA)和颈外动脉(ECA)，使用电凝器凝结ECA上甲状腺动脉和咽动脉并剪断。结扎ECA的远心端，并在其近心端挂线，暂时夹闭CCA和ICA，剪断ECA,将线栓从ECA残端插入ICA，结扎ECA残端，移除ICA的动脉夹，由ICA向内上方缓慢推进。适当调整方向，插入到线栓标记处(从CCA分叉处计算10mm)，开始计时，1h后移去线栓，观察无活动性出血后缝合切口。整个过程用电热毯保温，温度为36～37℃。将苏醒后的动物放回笼内，使其自由饮食。脑缺血24h，按Bederson Score评分法评估并记录神经功能缺损评分：0分为无功能障碍；1分为不能伸展右前肢；2分为向右侧旋转；3分为向右倾倒；4分为无自主活动伴意识障碍；5分为死亡。24h后麻醉剪下头部，取出脑组织，置于脑模具中(冰上)，沿视交叉向后切成2mm厚的切片，将其放入2％TTC的磷酸盐缓冲液，于37℃温箱中染色30min，再用4％多聚甲醛过夜固定并拍照。缺血体积百分数＝[缺血体积-(左半脑体积-右半脑体积)]/右半脑体积×100。缺血再灌注前10min，尾静脉给药1次，实验组分别为1、3、5mg/kg Sylvestin(根据实施例2合成)，对照组为生理盐水，体积100μl。缺血再灌注后10min内，尾静脉给药1次，剂量同上；6h再给药一次。每组6只。结果发现，Sylvestin可以有效减轻脑缺血再灌注造成的损伤(图2)。

Claims (10)

  1. 一种具有抗血栓活性的多肽，其衍生自森林山蛭抗血栓肽Sylvestin，其中所述多肽包含
    (1)与SEQ ID NO:1所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列，或
    (2)由SEQ ID NO:1所示氨基酸序列经添加、缺失、替换一个或更多个氨基酸得到的序列。
  2. 根据权利要求1的多肽，其包含如SEQ ID NO:1所示的氨基酸序列或由其组成。
  3. 根据权利要求1或2的多肽，其包含如SEQ ID NO:1所示的氨基酸序列，其中在SEQ ID NO:1所示氨基酸序列以外的氨基酸与天然森林山蛭抗血栓肽cDNA所编码氨基酸序列上相应位置的氨基酸保持一致。
  4. 根据权利要求1至3中任一项的多肽，其是基因工程生产的或者是化学合成的。
  5. 一种多核苷酸，其编码权利要求1至4中任一项所述多肽。
  6. 根据权利要求5的多核苷酸，其包含SEQ ID NO:2所示核苷酸序列中对应于SEQ ID NO:1所示氨基酸序列的核苷酸序列，例如其包含SEQ ID NO:2所示的核苷酸序列。
  7. 一种药物组合物，其包含根据权利要求1至4中任一项所述的多肽或根据权利要求5或6所述的多核苷酸，以及药学可接受的载体。
  8. 一种抑制FXIIa和/或kallikrein的方法，其包括将根据权利要求1至4中任一项所述的多肽、根据权利要求5或6所述的多核苷酸或者根据权利要求7所述的药物组合物暴露于FXIIa和/或kallikrein。
  9. 一种预防和/或治疗凝血所致疾病的方法，其包括：向有此需要的对象施用根据权利要求1至4中任一项所述的多肽，根据权利要求5或6所述的多核苷酸，或者根据权利要求7所述的药物组合物。
  10. 根据权利要求9的方法，其中所述凝血所致疾病是由血管内梗塞和/或血栓所致的疾病，例如选自动脉硬化、动脉粥样硬化、冠心病、中风、心肌梗塞、脑梗塞、脑缺血或急性脑缺血。
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