WO2011000263A1 - 姚虻纤维蛋白原水解酶tabfiblysin及其基因和应用 - Google Patents

姚虻纤维蛋白原水解酶tabfiblysin及其基因和应用 Download PDF

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WO2011000263A1
WO2011000263A1 PCT/CN2010/073987 CN2010073987W WO2011000263A1 WO 2011000263 A1 WO2011000263 A1 WO 2011000263A1 CN 2010073987 W CN2010073987 W CN 2010073987W WO 2011000263 A1 WO2011000263 A1 WO 2011000263A1
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tabfiblysin
yao
protease
horsefly
salivary gland
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PCT/CN2010/073987
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English (en)
French (fr)
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赖仞
马东莹
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中国科学院昆明动物研究所
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Priority to EP10793558A priority Critical patent/EP2450440A4/en
Priority to CN2010800301210A priority patent/CN103282091A/zh
Priority to US13/381,685 priority patent/US20120183525A1/en
Publication of WO2011000263A1 publication Critical patent/WO2011000263A1/zh

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    • 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)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6408—Serine endopeptidases (3.4.21)
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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
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6416—Metalloendopeptidases (3.4.24)
    • 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)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention provides a Yao fibrinogen hydrolase tabfiblysin and a gene and application thereof, and belongs to the field of biomedical technology. Background technique
  • Thrombotic diseases are a type of diseases that seriously affect human health, especially cardiovascular and cerebrovascular thromboembolic diseases, which have become the leading cause of death in our country.
  • Timely administration of antithrombotic drugs can greatly improve the survival rate of patients and reduce the extent and extent of organ damage.
  • Antithrombotic drugs mainly include thrombolytic drugs, anticoagulants and antiplatelet drugs.
  • Existing anticoagulants and anti-blood platelets have side effects such as hemorrhage, thrombocytopenia, and rebound after stopping the drug. Therefore, people are actively looking for new, highly effective, specific, and low-toxic antithrombotic drugs from various biological sources.
  • the cockroach insect commonly known as the horsefly, is an important arthropod and veterinary insect belonging to the arthropods of the order Diptera. Its adult is a kind of Chinese medicinal material, commonly known as aphids, which has the effect of promoting blood circulation and removing phlegm. It has a long history of medicinal use and can provide targets and inspiration for the development of modern new drugs. At the same time, the complexity of the traditional Chinese medicine ingredients and the limitations of the processing methods make the active ingredients of the medicines not function better. Therefore, finding specific active monomer compounds from these traditional medicines is one of the important contents of the modernization of traditional Chinese medicines. . Summary of the invention
  • the object of the present invention is to provide a Yao ⁇ fibrinogen hydrolase and a gene thereof and the technical solution of the present invention based on the prior art:
  • Yao ⁇ protease tabfiblysin is the first active protein we isolated from the salivary glands of Yao Yao, with a molecular weight of 2,7145.5 Daltons. Its full sequence primary structure is the amino acid sequence shown in SEQ ID NO: 2.
  • the cloning of the salivary gland protease tabfiblysin gene of Yaojing includes: total RA extraction from salivary glands, mR A purification, mRNA reverse transcription and cDNA library construction, primers were designed, and the coding gene was screened by PCR.
  • the result of gene sequencing revealed that the gene encoding the salivary gland protease tabfiblysin consisted of 768 nucleotides, and the sequence from the 5' end to the 3' end was the nucleotide sequence shown in SEQ ID NO: 1.
  • the Yaoqi salivary gland protease tabfiblysin of the present invention has a remarkable effect of hydrolyzing fibrinogen and inhibiting platelet aggregation, and can be used as a medicament for preparing a thrombotic disease.
  • Figure 1 shows Sephadex G-75 gel filtration chromatography of Yaojing salivary gland homogenate (SGE)
  • the arrow in the figure shows the active peak
  • Figure 2 is a diagram showing the SDS-PAGE analysis of the hydrolyzed fibrinogen of the present invention.
  • Figure 3 is the effect of tabfiblysin on ADP-induced human platelet aggregation.
  • Example 1 Cloning of the salivary gland protease tabfiblysin gene:
  • the supernatant was added with an equal volume of isopropanol, allowed to stand at room temperature for 10 minutes, centrifuged at 12,000 rpm for 10 minutes at 4 ° C, and the precipitate was washed once with 75% ethanol, and air-dried.
  • the sediment at the bottom of the tube was the total R A of the salivary gland of Yao.
  • Yao Yi salivary gland mR A was isolated and purified using PolyATtract® mRNA Isolation Systems from PROMEGA, USA.
  • B. Washing of magnetic beads (SA-PMP): Mix the magnetic beads with light bombs, adsorb to the magnetic frame for 30 seconds, discard the supernatant, add 0. 5x SSC 300 L, adsorb to the magnetic frame for 30 seconds, and finally add 100 L 0. 5xSSC suspension, called B liquid.
  • cDNA first strand synthesis (mRNA reverse transcription): 1. Add 1 ⁇ ⁇ salivary gland mRNA, 1 SMART IV primer, 1 CDS ⁇ /3' PCR primer in a sterile PCR tube, force B 2 ⁇ , deionized water to bring the total volume to 5 L.
  • the constructed cDNA contained approximately 1 x lO 6 individual clones.
  • NT3-F21 5'-Tac tec gg(g/c) gac aa(a/g) ct(g/a) cc(c/g) cgc-3';
  • CDSIII -primer 5'-att cta gag gcc gag gcg gcc atg-3 '
  • the reverse primer and the 5' end of the constructed cDNA library clone were forward-directed, and the forward and reverse primer sequences were:
  • NT3-R21 5'-(c/g)gg ( t/c)ag (c/t)tt gtc (g/c)cc gga gta-3'
  • PCR was carried out using the constructed Yaojing salivary gland cDNA as a template.
  • the reaction conditions were: pre-denaturation at 95 °C for 4 min, followed by 35 cycles at 94 ° C for 30 sec, 58 ° C for 30 sec, and 72 ° C for 40 sec. Then 72 ° C for 10 min.
  • a cDNA sequence encoding Tabfiblysin was inserted into the already constructed Yao ⁇ salivary gland cDNA library.
  • the sequencing results showed that the cDNA sequence encoding tabfiblysin consisted of 768 nucleotides, and the sequence from the 5' end to the 3' end was the nucleotide sequence shown in SEQ ID NO: 1.
  • Example 2 Preparation of Yaoqi salivary gland protease tabfiblysin:
  • the mixture was centrifuged at 1000 rpm/min for 10 min, and the supernatant was combined twice, lyophilized, and some samples were made to be water-soluble, desalted by Sephadex G-25, and lyophilized.
  • Each step of separation and purification is followed by activity monitoring using a hydrolyzed fibrinogen assay.
  • the first step Sephadex G-75 gel filtration: Yao ⁇ salivary gland homogenate lyophilized powder dissolved in 0.05 M Tris-HCl + 0.1M NaCl, pH 7.2 buffer, loaded on Sephadex-75 (Pharmacia products) coagulation A filter column (length 1000 mm, diameter 26 mm) was eluted with the same buffer to collect the active ingredient.
  • the second step FPLC Resource S ion exchange chromatography: Sephadex G-75 gel filtration of the active component peak (as shown in Figure 1), lyophilized in water, dialysis in 0.05 M Tris-HCl pH 8.3 buffer 12 Hr; loading on a Resource S column, eluting with a linear gradient of 0-0.5 M NaCl, and purifying the fibrinogen hydrolase.
  • This step is performed in the AKTA Purifier FPLC system.
  • Example 3 Pharmacological experiment of Yaoqi salivary gland protease:
  • Plasma-rich platelet aggregation (PRP) inhibition assay Healthy human platelets were diluted to 2.5 ⁇ 10 8 cells/mL with plasma. Take 300 ⁇ L of plasma-rich platelets, add appropriate amount of sample for 5 min, add ADP to induce aggregation, and record platelet aggregation for 5 min.
  • PRP Plasma-rich platelet aggregation
  • A the maximum aggregation that can be achieved by the agonist (this is the light transmittance, %);
  • B maximum aggregation that can be achieved with the addition of an agonist after the sample has been applied.
  • mice body weight 18-22 g, supplied by the Experimental Animal Center of Kunming Medical College
  • saline Take saline as a control.
  • the results showed that up to 18 ⁇ ⁇ / only dose of Tabfiblysin did not cause subcutaneous hemorrhage in mice.
  • Yaoqi salivary gland protease tabfiblysin has significant hydrolyzed fiber The role of proprotein and inhibition of platelet aggregation.
  • Yaoqi salivary gland protease tabfiblysin can be used as a drug for the preparation of thrombotic diseases.

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Abstract

本发明涉及一种姚虻唾液腺蛋白酶tabfiblysin及其基因和应用，属于生物医学领域。本发明的姚虻唾液腺蛋白酶分子量27145.5道尔顿。其全序列由255个氨基酸组成，其编码序列由768个核苷酸组成。本发明的姚虻唾液腺蛋白酶tabfiblysin具有显著的水解纤维蛋白原和抑制血小板聚集的作用，能作为制备治疗血栓性疾病药物的应用。

Description

姚虻纤维蛋白原水解酶 tabfiblysin及其基因和应用
技术领域
本发明提供一种姚虻纤维蛋白原水解酶 tabfiblysin及其基因和应用， 属于生物医学技 术领域。 背景技术
血栓性疾病是一类严重影响人类健康的疾病， 尤其是心脑血管血栓栓塞性疾病， 已成 为我国人口死亡的首要原因。 及时给予抗栓药物， 可大大提高病人的存活率， 减轻脏器损 害的程度和范围。 抗栓药物主要包括溶栓药、 抗凝药和抗血小板药。 现有的抗凝药和抗血 小板药存在着出血、 血小板减少及停药后反弹等副作用。 因此， 人们正在积极寻找着各种 生物来源的高效、 特异、 低毒的新型抗栓药物。
虻科昆虫俗称牛虻 （horsefly), 隶属于节肢动物门双翅目短角亚目虻科， 是重要的医 学和兽医学昆虫。 其成虫是一种中药材， 俗称虻虫， 具有活血化瘀， 破积痛经的功效。 其 药用历史悠久， 能为开发现代新药提供靶标和启示。 与此同时， 传统中药药物成分的复杂 性及其炮制方法的局限性造成药物活性成分不能更好的发挥作用， 因而从这些传统药物中 寻找特定的活性单体化合物是中药现代化的重要内容之一。 发明内容
本发明的目的旨在现有技术的基础上， 提供一种姚虻纤维蛋白原水解酶及其基因和应 用本发明的技术方案为：
姚虻蛋白酶 tabfiblysin是我们首次从姚虻唾液腺中分离得到的一种活性蛋白质， 分子 量 27145.5道尔顿。 其全序列一级结构为 SEQ ID NO: 2所示的氨基酸序列。
姚虻唾液腺蛋白酶 tabfiblysin基因的克隆包括： 姚虻唾液腺总 R A提取， mR A纯 化， mRNA反转录及 cDNA文库构建， 设计引物， 利用 PCR方法筛选编码基因。 基因测 序结果表明编码唾液腺蛋白酶 tabfiblysin基因由 768个核苷酸组成， 自 5'端至 3'端序列为 SEQ ID NO: 1所示的核苷酸序列。 本发明的姚虻唾液腺蛋白酶 tabfiblysin具有显著的水解纤维蛋白原和抑制血小板聚集 的作用， 能作为制备治疗血栓性疾病药物的被应用。 附图说明
图 1为姚虻唾液腺匀浆物 （SGE) 的 Sephadex G-75凝胶过滤层析
其中， 图中箭头所示为活性峰
图 2为本发明的水解纤维蛋白原作用 SDS-PAGE分析图
图 3为 tabfiblysin对 ADP诱导的人血小板聚集的影响 具体实施方式
实施例一： 姚虻唾液腺蛋白酶 tabfiblysin基因的克隆：
一、 姚虻唾液腺总 R A提取：
A.活体解剖姚虻（出自中国陕西省，学名 T. >^。 Macquart) ,挑取唾液腺立即放入液氮中， 迅速取一定量 （0.5-2 mg ) 唾液腺组织， 加入已预冷的 Trizol提取缓冲液 （美 国 Invitrogen公司产品） ， 置于冰上匀浆 15分钟。
B.加入 Trizol 1/5体积的氯仿， 剧烈混匀约 15秒， 室温放置 5分钟， 4 °C， 12000 rpm离心 10分钟， 取上清。
C.上清加入等体积的异丙醇， 室温放置 10分钟， 4°C， 12000 rpm离心 10分钟， 沉淀 用 75%乙醇洗一次， 晾干， 管底沉淀物即为姚虻唾液腺总 R A。
二、 姚虻唾液腺 mRNA的纯化：
姚虻唾液腺 mR A分离纯化采用美国 PROMEGA公司的 PolyATtract® mRNA Isolation Systems试齐 Ll盒。
A. 取姚虻唾液腺总 RNA 500 溶于 500 L DEPC水中， 65 °C水浴 10分钟， 加人 3 的 Oligo(dT) 探针和 13 L 20xSSC 溶液， 混匀， 放置室温冷却， 称为 A液。
B. 磁珠 (SA-PMP)的洗涤：将磁珠轻弹混匀，至磁力架吸附 30秒，弃上清，加 0. 5x SSC 300 L， 至磁力架吸附 30秒， 最后加 100 L 0. 5xSSC 悬浮， 称之为 B液。
C. 将 A液加入 B液中， 室温放置 10分钟， 至磁力架吸附 30秒， 弃上清， 用 0. l xSSC洗 涤 4次， 最后弃上清， 力口 lOO L DEPC水悬浮， 至磁力架上吸附 30秒， 将上清移至 新的试管， 再加入 150 L DEPC水重悬， 至磁力架吸附 30秒， 移上清至上述试管， 即为纯化的姚虻唾液腺 mRNA。
D. 加入 1/10体积 3 M乙酸钠， pH5. 2,等体积异丙醇， 于 -70 °C放置 30分钟， 4°C， 12000 rpm离心 10分钟， 弃上清， 沉淀溶解于 lO L DEPC水中。
三、 姚虻唾液腺 cDNA文库构建： 采用 CLONTECH公司 Creator™ SMART™ cDNA Library Construction Kit质粒 cDNA文库构建试剂盒。
A. cDNA第一链合成 （mRNA反转录)： 1. 在无菌 PCR管加入 1 姚虻唾液腺 mRNA、 1 SMART IV引物、 1 CDS ΙΠ/3'PCR引物， 力 B 2μ\,去离子水使总体积达到 5 L。
2. 混匀并短暂离心， 72°C 保温 2分钟。
3. 将离心管在冰上孵育 2分钟。
4. 在离心管中加入以下试剂 2.0 L5x第一链缓冲、 1.0 L20mMDTT、 l.O LlO mMdNTP混合物、 l.O LPowerScript逆转录酶。
5. 混合离心管中试剂并短暂离心， 在 42°C保温 1小时。
6. 将离心管置于冰上中止第一链的合成。
7. 从离心管取 2 L所合成的 cDNA第一链备用。
B. 采用长末端聚合酶链式反应 （LD-PCR) 方法扩增第二链
1、 95°C预热 PCR仪。
2、将 2 LcDNA第一链（mR A反转录）、 80 μΐ^去离子水、 10 μΐ^ 1 Ox Advantage 2 PCR缓冲、 2 L50xdNTP混合物、 2 5 'PCR引物、 2 CDS III/3' PCR引物 以及 2 L大肠杆菌聚合酶离心管进行反应。
3、 在 PCR仪中按以下程序扩增： 95°C， 20秒钟； 22个循环： 95°C， 5秒钟， 68°C，
6分钟。
4、 循环结束后， 将离心管中合成的 cDNA双链进行抽提。
C. 大肠杆菌 DH5ct感受态细胞的制备：
1. 挑取单个 DH5ct菌落 （购自北京天根生化科技有限公司）， 接种于 3mL不含氨苄 青霉素的 LB培养基中， 37°C培养过夜， 次日取上述菌液按比例 1:100再接种于 50 mLLB培养液中， 37°C振荡 2小时。 当 OD6QQ值达到 0.35时， 收获细菌培养物。 冰浴 10min， 于 4°C以 5000 rpm离心 10 min， 以回收细胞。
2. 每 mL初始培养液加 60(^L预冷的 0.1mol/LCaCl2-MgCl2溶液重悬。
3. 于 4°C以 5000 rpm离心 10 min， 以回收细胞。
4. 每 mL初始培养液加 60 预冷的 0.1mol/L CaCl2重悬细胞沉淀， 分装后备 用。
D. 酶切、 连接以及连接产物的转化：
1. 在微量离心管中加入 1 μί ρΜϋ19-Τ载体 （购自日本 Takara公司）、 4 姚虻 唾液腺 cDNA双链溶液， 全量为 5 L。
2. 加入 5 L (等量） 的连接酶缓冲混合物。
3.16°C反应 2小时。
4. 全量 （10 L) 加入至 100 μΐ, DH5a感受态细胞中， 冰浴 30分钟。
5.42°C加热 90秒钟后， 再在冰中放置 1分钟。
6. 加入 37°C温浴过的 LB培养基 900 L， 37°C缓慢振荡培养 60分钟。
7. 取 200 涂布于含有 X-Gal、 IPTG、 Amp的 LB培养基上 37°C培养 16小时， 形成单菌落。
8. 每个 LB平皿用 5 mL LB液体培养基洗涤菌落，加 30%甘油冻存。构建的 cDNA 大约含 l x lO6个单独克隆。
四、 姚虻唾液腺蛋白酶 tabfiblysin基因序列测定和结果：
根据分离纯化所得姚虻蛋白酶 tabfiblysin的内肽氨基酸序列和双翅目昆虫密码子的偏 嗜性， 我们设计了一条简并引物 NT3-F21 , 与构建姚虻唾液腺 cDNA文库时所使用的接头 引物 CDSIII配对， 正反向引物序列为：
NT3-F21： 5'-Tac tec gg(g/c) gac aa(a/g) ct(g/a) cc(c/g) cgc-3' ;
CDSIII -primer: 5'-att cta gag gcc gag gcg gcc atg-3 '
其中， 括号内的两个碱基表示简并引物。
以 NT3-F21和 3'PCR 为引物， 在我们已经构建好的姚虻唾液腺 cDNA文库中筛选插入 了编码 tabfiblysin的 cDNA序列的克隆。
根据测得的成熟蛋白的核苷酸序列设计反向引物和所构建的 cDNA文库克隆的 5'端保 守序列为正向引物， 正反向引物序列分别为：
5 'primer: 5 '-aag cag tgg tat caa cgc aga gt-3 '
NT3-R21 : 5'-(c/g)gg ( t/c)ag (c/t)tt gtc (g/c)cc gga gta-3'
以构建的姚虻唾液腺 cDNA为模板， 进行 PCR。 其反应条件为： 95 °C预变性 4 min， 接 着在如下条件进行 35轮循环， 94°C 30 sec， 58°C 30 sec, 72°C 40 sec。 然后 72°C 10 min。 通 过上述方法， 在已经构建好的姚虻唾液腺 cDNA文库中筛选插入了编码 Tabfiblysin 的 cDNA序列。 测序结果表明编码 tabfiblysin的 cDNA序列由 768个核苷酸组成， 自 5'端至 3' 端序列为 SEQ ID NO: 1所示的核苷酸序列。 实施例二： 制备姚虻唾液腺蛋白酶 tabfiblysin :
一、 姚虻唾液腺的解剖，匀浆与处理
姚虻 (中国陕西省， 学名 r. y^ Macquart) )解剖的方法如下： 用小剪刀将虫体沿背中线剪 开， 放入 4°C含 0.15M NaCl， pH7.2的 0.01M的磷酸盐缓冲液中浸泡， 挑取唾液腺， 放入含 上述缓冲液的烧杯中存于 -20 °C，解剖完毕后 10000 rpm离心 10 min,上清继续存放于 -20 °C， 沉淀用玻璃匀浆器在冰上充分匀浆后 lOOOO rpm/min离心 10 min， 合并两次离心上清， 冻干 并取部分样品水溶后用 Sephadex G-25脱盐，冻干。
二、 分离纯化的每一个步骤都用水解纤维蛋白原检测方法进行活性跟踪。 第一步， Sephadex G-75凝胶过滤： 姚虻唾液腺匀浆液冻干粉溶解于 0.05 M Tris-HCl + 0.1M NaCl, pH7.2的缓冲液， 上样于 Sephadex -75( Pharmacia产品)凝胶过滤柱 (长 1000 mm, 直径 26 mm)， 用同样缓冲液洗脱， 收集活性成分。
第二步， FPLC Resource S 离子交换层析： Sephadex G-75 凝胶过滤得到的活性成分峰 (如图 1所示），冻干溶解于水，于 0.05 M Tris-HCl pH 8.3 缓冲液透析 12 hr;上样于 Resource S柱， 用一个 0-0.5M NaCl 的线性梯度进行洗脱， 纯化得到纤维蛋白原水解酶。该步操作在 AKTA Purifier FPLC系统中进行。 实施例三： 姚虻唾液腺蛋白酶的药理实验：
一、 纤维蛋白原水解活性检测
取 10 浓度为 2 mg/mL的纤维蛋白原（购自美国 Sigma公司，溶于含有 150 mM NaCl 的 50 mM Tris-HCl， pH7.4缓冲液） 与样品于 37°C保温 24 hr后， 进行 12% SDS-PAGE还 原电泳分析， 如图 2所示。 实验结果发现此酶能明显地水解纤维蛋白原的（X-链， 而对 β- 链的水解活性低， 不能水解 γ链， 其水解活性排序为 α > β > γ。
二、 血小板聚集抑制实验
富血浆小板聚集 （PRP ) 抑制实验： 健康人血小板用血浆稀释至 2.5X 108 个 /mL。 取 富血浆血小板 300 μL, 加入适量样品保温 5min后， 添加 ADP诱导聚集， 记录 5 min血 小板聚集情况。
聚集抑制率 （I) 的计算：
Figure imgf000006_0001
A=激动剂所能达到的最大聚集 （本文为透光率， ％);
B=样品作用后加入激动剂所能达到的最大聚集。
结果表明， Tabfiblysin能够明显的抑制 ADP诱导的血小板聚集， 且抑制率随抑制剂浓 度的增大而升高 （如图 3所示）。
三、 出血活性检测
将样品溶解到生理盐水中， 使其浓度为 0.6 mg/mL。取 30 μΐ对 ICR小鼠（体重 18-22 g， 昆明医学院实验动物中心提供） 进行皮下注射， 24 小时后， 剥离小鼠皮肤， 观察内皮 出血斑的大小。 以生理盐水做对照。 结果表明高达 18 μ§/只的剂量的 Tabfiblysin都不引起 小鼠皮下出血。
姚虻唾液腺蛋白酶的药理实验表明： 姚虻唾液腺蛋白酶 tabfiblysin具有显著的水解纤 维蛋白原和抑制血小板聚集的作用。 姚虻唾液腺蛋白酶 tabfiblysin能作为制备治疗血栓性 疾病药物的被应用。

Claims

权 利 要 求
1、 姚虻蛋白酶 tabfiblysin, 其特征在于姚虻蛋白酶 tabfiblysin是从姚虻唾液腺中分离 得到的一种活性蛋白质， 分子量 27145.5道尔顿， 其全序列一级结构为 SEQ ID NO: 2所示 的氨基酸序列。
2、 姚虻蛋白酶 tabfiblysin基因， 其特征在于该基因由 768个核苷酸组成， 自 5'端至 3'端 序列为 SEQ ID NO: 1所示的核苷酸序列。
3、 姚虻蛋白酶 tabfiblysin的应用， 其特征在于姚虻蛋白酶 tabfiblysin能水解纤维蛋白 原和抑制血小板聚集， 且无出血活性， 能作为制备治疗血栓性疾病药物的应用。
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