WO2010034176A1 - 来自尖吻蝮蛇的类凝血酶 - Google Patents

来自尖吻蝮蛇的类凝血酶 Download PDF

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WO2010034176A1
WO2010034176A1 PCT/CN2009/000230 CN2009000230W WO2010034176A1 WO 2010034176 A1 WO2010034176 A1 WO 2010034176A1 CN 2009000230 W CN2009000230 W CN 2009000230W WO 2010034176 A1 WO2010034176 A1 WO 2010034176A1
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pbs
solution
ultrafiltration
snake venom
enzyme
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孙狄
王锡娟
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HEALTHSTAR PHARMACEUTICAL CO Ltd
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Priority to EP09815546.8A priority Critical patent/EP2343370B8/en
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Priority to HK11112050.9A priority patent/HK1157816B/zh
Priority to US13/121,080 priority patent/US8476054B2/en
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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes

Definitions

  • the present invention relates to a serine protease, in particular to a snake venom hemagglutinase, and to a method for its isolation and purification. Background technique
  • TLC thrombin-like enzyme
  • thrombin-like enzymes act similarly to thrombin (thrombin), which converts fibrinogen in plasma into fibrin and "coagulates". It has been found so far that more than 30 kinds of snake venom contain thrombin-like components, and more than 20 kinds have been isolated and purified, and all or part of the amino acid sequence of more than 10 kinds of thrombin has been elucidated.
  • the molecular weight of TLC has been found to be between 29 and 45 kD, most of which are acidic glycoproteins.
  • Rhilase is a thrombin isolated from the snake venom of Bothmps atrox, which consists of 255 amino acids with a guide peptide of 24 amino acids at the N-terminus.
  • the active enzyme contains 231 amino acids and has a relative molecular weight of 39 to 43 kD, which is a single-chain glycoprotein.
  • thrombin-like enzymes Two "thrombin-like enzymes" are isolated from Agkistrodon acutus, which are double-stranded molecular structures.
  • the A subunit of thrombin I contains 132 amino acids with a molecular weight of 16kD; the B subunit contains 123 amino acids, The amount of enzyme is 14 kD, and the specific activity of the enzyme is: 160 u/mg.
  • the A subunit of thrombin oxime contains a basal acid with a molecular weight of 15 kD; the B subunit contains 120 amino acids with a molecular weight of '13 kD and an enzyme specific activity of 70 u/mg.
  • the two TLCs have been proved by pharmacological experiments: they all have hemostatic effects.
  • a highly active hemagglutinase is isolated from the snake venom of Agkistrodon acutus (commonly known as the five-step snake). Summary of the invention
  • the hemagglutinase of the present invention is a highly active hemagglutinase which is isolated from the Chinese cockroach ⁇ Agkistrodon acutus and snake venom.
  • the enzyme has the following characteristics: 1
  • the enzyme protein contains 252 amino acids with a molecular weight of 29.3 to 29.5 kD and an isoelectric point of pi of 5.5. 2 is composed of two subunits ⁇ and ⁇ , and the subunit chains are connected by seven disulfide bonds.
  • the ⁇ subunit contains 129 amino acids, has a molecular weight of 15 kD, and has an amino acid sequence as shown in SEQ ID No. 1.
  • the ⁇ subunit contains 123 amino acids and has a molecular weight of 14.5 kD, and the amino acid sequence thereof is shown in SEQ ID No. 2.
  • 4 Enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating that it is a serine protease. 5 can hydrolyze human fibrinogen alpha chain.
  • PMSF phenylmethylsulfonyl fluoride
  • the present invention also provides a method for purifying the above hemagglutinase, which comprises the following steps:
  • the above eluate is appropriately concentrated, dialyzed or subjected to multiple dilutions of ultrafiltration to remove NaCl;
  • the dialysis solution was again applied to a pre-equilibrated DEAE-Sephrose FF column, washed with 0.01 M PBS pH 7.0 ⁇ 7.5, and then 0.01 M with 0 ⁇ 1 M NaCl
  • the PBS was partially eluted with pH 7.0 - 7.5, and the second elution peak in the eluate of 0.06 M NaCr solution was collected;
  • step 1) snake venom pretreatment method is to dissolve the snake venom with an appropriate amount of pre-cooled 0.01M PH7.0 - 7.5 PBS, centrifuge to take the supernatant for dialysis (dialysis bag molecular weight cutoff is 7,000D ⁇ 10,000D) or The centrifugation supernatant was repeatedly diluted and concentrated by ultrafiltration using a tangential flow ultrafiltration method (ultrafiltration membrane molecular weight cutoff of 5,000 D to 10,000 D). Pretreatment can remove insoluble impurities as well as small molecule peptides and reduce solution ionic strength.
  • the pretreatment of the snake venom can be carried out by the following steps: weighing a few grams of snake venom, using a venom weight of 5 to 10 times the volume of pre-cooled 0.01 M ⁇ ⁇ 7.0 ⁇ 7.5 PBS in a 4 to 8 ° C chromatography cabinet Stir and dissolve for 30 ⁇ 60 minutes, centrifuge at 4 ⁇ 8°C, 5,000 ⁇ 10,000g for 10 ⁇ 20 minutes, pour the supernatant into the dialysis bag, add the venom weight again and add 5 ⁇ 10 times volume pre-cooling. The PBS was stirred and suspended, and centrifuged again.
  • each dialysis step can be replaced by tangential flow ultrafiltration (ultrafiltration membrane molecular weight cutoff of 5,000 ⁇ 10000D), diluted by PBS, concentrated by ultrafiltration, diluted, and then concentrated by ultrafiltration. Removal of small molecule polypeptides and desalting reduces solution ionic strength.
  • step 2) and step 4) can be used to pre-balance the DEAE-Sephrose FF column with 0.01M PBS pH 7.0 ⁇ 7.5, and then load.
  • step 3) and step 5) are to remove the NaCl present in the solution.
  • the eluent is concentrated by ultrafiltration.
  • the small volume concentrate can be directly dialyzed; it can also be concentrated by PBS, concentrated by ultrafiltration, diluted, and concentrated by ultrafiltration to concentrate the protein and remove the NaCl.
  • the final purified enzyme concentrate can be desalted directly using a Sephadex-G25 column.
  • the salt-removed solution is directly lyophilized or freeze-dried by adding a lyoprotectant.
  • a lyoprotectant may be low molecular weight dextran, or mannitol, sucrose, glycerin, gelatin or human serum albumin, and the like.
  • the cryoprotectant is added in an amount of from 1% to 5% (w/v).
  • the hemagglutinase purified by the method of the invention has a specific activity of not less than 180 u/mg protein, polyacrylamide gel electrophoresis (PAGE)-band, reduction SDS-polyacrylamide gel electrophoresis (reduced SDS-PAGE) HPLC analysis of purity above 95%. Based on the weight of the snake venom raw material, the purification yield of this method is 0.7% - 0.8%.
  • the hemagglutinase of the invention has good agglutination activity and can be prepared into various hemostatic drugs, for example, by appropriately diluting to a prescribed enzyme living unit, and then adding a lyoprotectant (low molecular weight dextran 20 or human serum albumin, etc.), by virus Membrane filtration, freeze-drying to make medical injection freeze-dried powder needles, used for hemostasis in surgery, and various clinical bleeding symptoms. It can also be used as a wound external hemostatic patch, powder or liquid spray.
  • Figure 1 shows the hydrolysis of human fibrinogen by the hemagglutinase of the present invention, 4h, 2h, lh, 0.5h: respectively representing the time when hemagglutinin hydrolyzes human fibrinogen; F: human fibrinogen control; M: Protein molecular weight standard.
  • the percent sign "%" referred to in the present invention means mass percentage unless otherwise specified; however, the percentage of the solution, unless otherwise specified, means that the solution contains several grams of solute in 100 ml; the percentage between the liquids, It refers to the ratio of capacity at 20 °C.
  • the pretreated snake venom solution was pre-equilibrated with 0.01 M pH 7.4 PBS on a DEAE-Sepharose Fast Flow anion exchange chromatography column, washed with 0.01 M pH 7.4 PBS, and then 0.02 M, 0.06 M and 1.0 M NaCl, respectively. 0.01 M pH 7.4 PBS was eluted in sections, and the elution peak of 0.06 M NaC solution was collected;
  • the dialyzed enzyme solution was pre-equilibrated on a DEAE-Sepharose Fast Flow anion exchange chromatography column, washed with 0.01 M pH 7.4 phosphate buffer, and then 0.02 M, 0.04 M, 0.06 M, and 1.0 M NaCl, respectively.
  • 0.01 M pH 7.4 PBS was eluted in sections, and a second elution peak of the 0.06 M NaCl solution eluate was collected to obtain 1050 ml of an eluent. It was confirmed by enzyme activity measurement that the PAGE was a band indicating that it had been purified.
  • the solution was concentrated to 135 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane).
  • the 135 ml concentrate was placed in a dialysis bag, and dialyzed against ion-free water for 24 hours, during which time the solution was changed 3 times, 5 liters each time.
  • the volume after dialysis was 159 ml, and the total protein content in the solution was determined to be 240 mg, and the specific activity of the enzyme was determined to be 195 u/mg protein, and the final yield was 0.8%.
  • the purity of the HPLC analysis was 98.2%, and the reduced SDS-PAGE was two bands with molecular weights of approximately 15 kD and 14.5 kD, respectively.
  • the isoelectric point pi of the enzyme was determined by isoelectric focusing electrophoresis to be 5.5.
  • the amino acid sequences of the two bands are shown by SEQ ID No. 1 and SEQ ID No. 2, respectively, by amino acid sequencing by DENOVO method.
  • the alpha subunit (SEQ ID No. 1) contains 129 amino acids, and the molecular weight calculated by amino acid is only 14660.7Dalton;
  • the ⁇ subunit (SEQ ID No. 2) contains 123 amino acids, and the molecular weight calculated by amino acid is only 14551.8 Dalton.
  • Both the alpha subunit and the beta subunit contain seven cysteine residues.
  • the two centrifugation supernatants were combined in a dialysis bag (molecular weight cutoff of 10000 D), dialyzed against 0.01 M pH 7.4 PBS for 24 hours in a 4 °C chromatography cabinet, and the solution was changed three times.
  • the pretreated snake venom solution was subjected to the first DEAE-Sephrose FF column chromatography in the same manner as in Example 1, and the target substance appeared in the elution peak of 0.06 M NaCl by enzyme activity measurement and electrophoresis.
  • the collected solution was obtained in a total of 2262 ml, and concentrated to 200 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane), and the 200 ml ultrafiltration concentrate was poured into a dialysis bag (10,000 D).
  • the solution was dialyzed against 5000 ml of 0.01 M pH 7.4 PBS at 4 ° C for 24 hours, during which time the solution was changed 3 times.
  • the dialyzed enzyme solution was applied to a DEAE-Sephrose FF column, and a second chromatography was carried out in the same manner as in Example 1. Hemagglutinase appeared in the second elution peak of the 0.06 MnaCr solution eluate, and 956 ml of the peak eluate was collected. It was confirmed by enzyme activity measurement that PAGE was a band indicating that it had been purified.
  • the solution was concentrated to 192 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane).
  • the 192 ml of the concentrated solution was applied to a Sephdex-G25 column, and the column was desalted by washing with ion-free water to collect 322 ml of the eluted peak.
  • the solution was directly freeze-dried to obtain a total protein of 235 mg, and the specific activity of the enzyme was determined to be 200 u/mg protein. The rate is 0.78%.
  • Example 3 Purification of snake venom hemagglutinase
  • the target protein appeared in the eluent of 0.06 M NaCl solution, and a total of 6750 ml was collected from the eluted peak.
  • the collected liquid was concentrated to 500 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 8 k membrane).
  • the enzyme activity assay confirmed that the target protein appeared in the second elution peak of the 0.06 M NaCl solution eluate, and the elution peak collected a total of 3050 ml.
  • the PAGE was checked as a strip indicating that it had been purified.
  • the filtrate is directly dispensed and then freeze-dried.
  • Example 2 The hemagglutinase isolated in Example 2 was further purified by HPLC to obtain a solution of 100 mg of the protein of 100 mg/mL.
  • the enzyme activity was 10 u/mL.
  • bovine fibrinogen (Sigma) was prepared in physiological saline.
  • the phenylmethylsulfonyl fluoride (PMSF, Merck) was dissolved in isopropanol at a solution concentration of 4 mg/ml.
  • the agglutination test was separately performed in the order of the test tube numbers. Add a constant temperature of 1% bovine fibrinogen solution 200 ⁇ 1 to the test tube, immediately time it, mix gently while shaking, and let stand in water at 37 °C to observe the agglutination reaction in the test tube. The timing is terminated when the solution is completely solidified.
  • a 4 mg/ml human fibrinogen solution was prepared in 50 mM Tris-HCl (pH 7.4) buffer.
  • 0.5 ml of human fibrinogen solution was added to each of 5 small test tubes, and one active unit of hemagglutinase was added to 4 tubes at intervals of 4 different hydrolysis incubation times. The temperature was maintained at 37 °C for 4, 2, and 0.5 hours. Immediately after the incubation, SDS-PAGE electrophoresis was performed to observe the degree of hydrolysis of human fibrinogen.
  • Another 0.5 ml fibrinogen control tube was incubated for 4 hours in a 37 ° C water bath.
  • Bovine fibrinogen assay 1.0% bovine fibrinogen (Sigma) solution prepared in physiological saline was placed in a small test tube, kept in a 37 ⁇ 0.5°C water bath for 3 minutes, and preheated at 37 ⁇ 0.5°C. The enzyme solution is 1 ml, immediately timed, and the fibrinogen solution shakes white flocs in 120 seconds and 30 seconds, and the enzyme solution is lu/ml.
  • Standard human plasma assay Take 1 ml of standard human plasma in a small test tube, preheat for 3 minutes in a 37 ° C ⁇ 0.5 ° C water bath, add 37 ⁇ 0.5 ° C preheated enzyme solution 1 ml, immediately timed, human Plasma within 60 ⁇ 20 seconds When the white floc appears in the shaking, the enzyme solution is lu/ml.
  • the dilution factor is the number of enzyme activity units per ml of the original enzyme solution.

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Description

来自尖吻蝮蛇的类凝血酶 技术领域
本发明涉及一种丝氨酸蛋白酶， 具体地说是一种蛇毒血凝酶， 本 发明还涉及其分离纯化方法。 背景技术
据国内外文献的报导， 在蝮亚科 （Crotalinae ) 蛇毒中多存在一 类与血液凝固相关的蛋白酶， 通常称之为 "类凝血酶"（thrombin-like enzyme, 简称： TLC )。 类凝血酶与凝血酶 （thrombin ) 的作用相似， 可以使血浆中纤维蛋白原转化为纤维蛋白而 "凝固"。 迄今已经发现 现已在 30多种蛇毒中含有类凝血酶成分,并有 20余种得到分离纯化， 其中有 10余种类凝血酶的全部或部分氨基酸序列得到阐明。 已发现 的 TLC分子量多在 29 ~ 45kD之间， 大多数为酸性糖蛋白。
在以往已经发现的蛇毒 TLC 中， 蛋白质一级结构多为单链。 其 代表产品 "立苴雪" （ Reptilase )是由巴西矛头蝮蛇 （ Bothmps atrox ) 蛇毒中分离出的凝血酶， 该酶前体由 255个氨基酸构成， N端有 24 个氨基酸形成的引导肽，活性酶含 231氨基酸，相对分子量 39 ~ 43kD， 为单链糖蛋白。
近 10年来的研究发现在蝮亚科蛇毒 TLC 中也存在双链分子结 构， 链间由二硫键连接。 Xin Cheng等（中国科技大学） 1999年报导： 从五步蛇 gkistrodon acutus ) 中分离到一种 "类凝血酶"， 命名为 "Agkisacutacin"。 该蛋白由两条肽链构成， ct -亚基分子量 15kD， （3 -亚基分子量 14kD。 Agkisacutacin能够水解纤维蛋白原中的 α链。 肖 昌华（中科院昆明动物研究所） 2004年从五步蛇（ Agkistrodon acutus ) 中分离到两种 "类凝血酶"， 均为双链分子结构。 凝血酶 I的 A亚基 含有 132个氨基酸， 分子量 16kD; B亚基含有 123个氨基酸， 分子 量 14kD, 酶比活性为： 160u/mg。 凝血酶 Π的 A亚基含有. 个 基酸， 分子量 15kD; B亚基含有 120个氨基酸， 分子量 ' 13kD， 酶比 活性为： 70u/mg。 该两种 TLC经药理实验证明： 均具有止血功效。
我国具有丰富的蛇毒研究资源， 本发明人从我国的尖吻蝮蛇
(Agkistrodon acutus, 俗称五步蛇）的蛇毒中分离得到一种高活性血凝 酶。 发明内容
本发明的目的在于提供一种蛇毒血凝酶，其是从尖吻蝮蛇蛇毒中 分离得到的一种类凝血酶。
本发明的另一个目的在于提供一种分离纯化上述血凝酶的方法。 本发明血凝酶是从中国尖吻蝮蛇 ί Agkistrodon acutus、蛇毒中分 离得到的高活性血凝酶。 该酶具有如下特征： ①酶蛋白含 252个氨基 酸， 分子量为 29.3〜29.5kD， 等电点 pi为 5.5。 ②由 α、 β两个亚基构 成， 亚基链间由七个二硫键连接。 ③ α亚基含 129个氨基酸， 分子量 为 15kD,其氨基酸序列如 SEQ ID No. l所示； β亚基含 123个氨基酸， 分子量为 14.5kD，其氨基酸序列如 SEQ ID No.2所示。④酶活性可被 苯甲基磺酰氟（PMSF)完全抑制， 表明其是一种丝氨酸蛋白酶。 ⑤能 够水解人纤维蛋白原 α链。
本发明还提供上述血凝酶的纯化方法， 其包括如下步骤：
1 )、 蛇毒预处理；
2 )、 将预处理后的蛇毒溶液上经预平衡的 DEAE - Sephrose FF 阴离子交换层析柱，用 0.01M pH7.0 ~ 7.5的 PBS洗柱， 再用含 0〜1M NaCl的 0.01M pH7.0 ~ 7.5的 PBS分段洗脱， 收集 0.06M NaCl溶液 的洗脱液；
3 )、将上述洗脱液适当浓缩后透析或经多次稀释超滤去除 NaCl;
4 )、 将透析后的溶液再次上经预平衡的 DEAE - Sephrose FF层 析柱，用 0.01M pH7.0 ~ 7.5 的 PBS洗柱，再用含 0~1M NaCl的 0.01M pH7.0 - 7.5 的 PBS分段洗脱，收集 0.06M NaCr溶液洗脱液中的第二 个洗脱峰；
5 )、将上述洗脱液适当浓缩后用蒸馏水透析或采用 Sephadex-G25 柱脱盐。
其中， 步骤 1 )蛇毒预处理的方法是将蛇毒用适量预冷的 0.01M PH7.0 - 7.5 PBS溶解， 离心取上清液进行透析 (透析袋截留分子量为 7，000D~10，000D)或釆用切向流超滤 （超滤膜截留分子量为 5，000D ~ 10,000D )方法， 将离心上清液反复多次稀释、 超滤浓缩。 通过预处 理可以去除不溶的杂质以及小分子多肽， 并降低溶液离子强度。
具体地说可通过如下步骤进行蛇毒的预处理： 称取蛇毒若干克， 用蛇毒重量 5〜10倍体积预冷的 0.01M ρΗ7.0 ~ 7.5 的 PBS于 4〜8°C的 层析柜中搅拌溶解 30~60分钟， 于 4~8°C、 5，000~10,000g离心 10〜20 分钟，将离心上清液倾入透析袋中，离心沉淀再次加入蛇毒重量 5〜10 倍体积预冷的 PBS搅拌悬浮， 再次离心。 合并两次离心上清液于透 析袋 (截留分子量为 7，000D〜10，000D)中， 在 4~8°C层析柜中对 0.01M pH7.0 ~ 7.5的 PBS透析 12〜24小时， 期间换液 2〜4次， 以去除小分 子多肽和降低溶液离子强度。
如上所述， 各透析步骤均可釆用切向流超滤（超滤膜截留分子量 为 5，000~10000D )方法代替， 通过 PBS稀释、 超滤浓缩， 再稀释、 再超滤浓缩的方式以去除小分子多肽和脱盐降低溶液离子强度。
其中， 步骤 2 )和步骤 4 )可釆用 0.01M pH7.0 ~ 7.5 的 PBS预平 衡 DEAE - Sephrose FF层析柱， 然后上样。
其中， 步驟 3 )和步骤 5 ) 的目的均是去除溶液中存在的 NaCl。 洗脱液浓缩方式为超滤浓缩。 小体积浓缩液可直接进行透析； 也可通 过 PBS稀释、 超滤浓缩， 再稀释、 再超滤浓缩的方式以浓缩蛋白， 脱去 NaCl。最终被纯化的酶浓缩液可直接釆用 Sephadex-G25柱脱盐。
除盐后的溶液直接冷冻干燥， 或加入冻干保护剂冷冻干燥。 所述 冻干保护剂可以是低分子右旋糖酐、 或甘露醇、 蔗糖、 甘油、 明胶或 人血白蛋白等等。 冷冻保护剂的加入量为 l% - 5%(w/v)。
经本发明方法纯化的血凝酶比活力不低于 180u/mg蛋白，聚丙烯 酰胺凝胶电泳 ( PAGE ) —条带， 还原 SDS-聚丙烯酰胺凝胶电泳（还 原 SDS-PAGE )两条带； HPLC分析纯度 95%以上。 以蛇毒原料重量 计， 本法纯化收得率为 0.7% - 0.8%。
本发明血凝酶具有良好的凝集活性， 可制成各种止血药物， 例如 经适当稀释到规定酶活单位， 再添加冻干保护剂 （低分子右旋糖酐 20或人血白蛋白等）， 经病毒膜过滤， 冷冻干燥制成医用注射冻干粉 针， 用于外科手术中止血， 以及各种临床出血症状。 也可制成创伤外 用止血贴剂、 粉剂或液体喷雾剂。 附图说明
图 1 显示的是本发明血凝酶对人纤维蛋白原的水解作用， 4h、 2h、、 lh、 0.5h: 分别代表血凝酶水解人纤维蛋白原的时间； F: 人纤 维蛋白原对照； M: 蛋白质分子量标准物。 具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的 限制。 在不背离本发明精神和实质的情况下， 对本发明方法、 步骤或 条件所作的修改或替换， 均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟 知的常规手段。
本发明中涉及到的百分号 "％"， 若未特别说明， 是指质量百分 比； 但溶液的百分比， 除另有规定外， 是指溶液 100ml中含有溶质若 干克； 液体之间的百分比， 是指在 20°C时容量的比例。 本发明中类 似 "用蛇毒重量 10倍体积预冷的" 表述， 其中的重量和体积的单位 分别是 g和 ml。 实施例 1 蛇毒血凝酶的纯化
取 30g尖吻蝮蛇蛇毒干粉（批号： 20061001， 广西蛇毒研究所）， 用蛇毒重量 10倍体积预冷的 0.01M pH7.4 的 PBS于 4°C的层析柜中 搅拌溶解 30分钟， 于 4°C、 lOOOOg离心 10分钟， 将离心上清液倾入 透析袋中， 离心沉淀再次加入蛇毒重量 10倍体积预冷的 PBS搅拌悬 浮， 再次离心。 合并两次离心上清液于透析袋 (截留分子量为 7000D) 中，在 4°C层析柜中对 0.01M pH7.4的？88透析24小时，期间换液 3 次。 将预处理后的蛇毒溶液上经 0.01M pH7.4PBS预平衡的 DEAE - Sepharose Fast Flow阴离子交换层析柱， 用 0.01M pH7.4PBS洗柱， 再分别用 0.02M、 0.06M和 1.0M NaCl的 0.01M pH7.4的 PBS分段 洗脱， 收集 0.06M NaC 溶液的洗脱峰；
经酶活测定（参照附录①或⑦方法）和电泳分析， 目的物出现在 0.06M NaC 溶液的洗脱峰中， 合并洗脱收集液， 共得 2220ml， 釆用 Millipore Pellicon 2 切向流超滤器 ( 0.1M2 cut off 5k膜 )超滤浓缩至 200ml, 将该 200ml 超滤浓缩液倾入透析袋（7000D)中， 用 5000ml 0.01M pH7.4的 PBS于 4°C透析 24小时，期间更换溶液 3次。将透析 后的酶溶液上经预平衡的 DEAE - Sepharose Fast Flow阴离子交换层 析柱， 用 0.01M pH7.4磷酸缓冲液洗柱， 再分别用 0.02M、 0.04M、 0.06M和 1.0M NaCl的 0.01M pH7.4的 PBS分段洗脱， 收集 0.06M NaCl溶液洗脱液的第二个洗脱峰， 得洗脱液 1050ml。 经酶活测定确 认， PAGE检查为一条带，说明已经得到纯化。釆用 Millipore Pellicon 2 切向流超滤器（0.1M2 cut off 5k膜）超滤浓缩至 135ml。将该 135ml 浓缩液装入透析袋中， 对无离子水进行透析 24小时， 期间更换溶液 3次， 每次 5升。 透析后体积为 159ml, 测定该溶液中总蛋白质含量 为 240mg， 测定酶比活力为 195u/mg蛋白， 最终收率 0.8%。 HPLC 分析纯度 98.2 %， 还原 SDS - PAGE为两条带， 其分子量分别大致为 15kD和 14.5kD。 等电聚焦电泳测定该酶等电点 pi为 5.5。 经 DENOVO法氨基酸测序， 这两条带的氨基酸序列分别如 SEQ ID No.l和 SEQ ID No.2所示。 α亚基（SEQ ID No.l )含 129个氨基 酸， 仅按氨基酸计算分子量为 14660.7Dalton; β亚基（ SEQ ID No.2 ) 含 123个氨基酸， 仅按氨基酸计算分子量为 14551.8Dalton。 α亚基和 β亚基均各含有 7个半胱氨酸残基。 实施例 2 蛇毒血凝酶的纯化
取 30g尖吻蝮蛇蛇毒干粉（批号： 20061001， 广西蛇毒研究所）， 用 300ml预冷的 0.01M pH7.4 的 PBS于 4°C的层析柜中搅拌溶解 60 分钟， 于 4°C、 10000g离心 15分钟， 将离心上清液倾入透析袋中， 离心沉淀再次加入 300ml预冷的 PBS搅拌悬浮， 再次离心。 合并两 次离心上清液于透析袋 (截留分子量为 10000D)中， 在 4°C层析柜中对 0.01M pH7.4的 PBS透析 24小时， 期间换液 3次。
按与实施例 1 相同的方法， 将经预处理的蛇毒溶液进行第一次 DEAE-Sephrose FF 柱层析， 经酶活测定和电泳分析目的物出现在 0.06MNaCl的洗脱峰中，合并洗脱收集液，共得 2262ml，釆用 Millipore Pellicon 2 切向流超滤器（ 0.1M2 cut off 5k膜）超滤浓缩至 200ml， 将 该 200ml 超滤浓缩液倾入透析袋（10,000D)中， 用 5000ml 0.01M pH7.4PBS于 4°C透析 24小时， 期间更换溶液 3次。 透析后的酶液上 样至 DEAE-Sephrose FF柱中， 按适实例 1相同的方法进行第二次层 析。血凝酶出现在 0.06MnaCr溶液洗脱液的第二个洗脱峰中， 收集该 峰洗脱液得 956ml。 经酶活测定确认， PAGE检查为一条带， 说明已 经得到纯化。釆用 Millipore Pellicon 2 切向流超滤器（ 0.1M2 cut off 5k 膜）超滤浓缩至 192ml。将该 192ml浓缩液上样至 Sephdex-G25柱上， 用无离子水洗柱脱盐， 收集洗脱峰 322ml， 该溶液直接冷冻干燥后收 获总蛋白 235mg， 测定酶比活力为 200u/mg蛋白， 最终收率 0.78%。
HPLC分析纯度 99 %， 色谱图与实施例 1相一致， 还原 SDS - PAGE 为两条带， 其分子量分别大致为 15kD和 14.5kD。 实施例 3 蛇毒血凝酶的纯化
1、 称取 100克尖吻蝮蛇蛇毒干粉（批号 20061102 )， 广西蛇毒 研究所）用 2000ml预冷的 0.01M pH7.4 的 PBS于 4 - 8°C的层析拒 中搅拌溶解 60分钟， 4°C 10000g离心 20分钟， 将离心上清液倾入 一个 3000ml烧杯中， 釆用 Millipore Pellicon 2 切向流超滤器（0.1M2 cut off 8k膜）超滤浓缩至 500ml， 再加入预冷的 1500ml 0.01M pH7.4 的 PBS， 再超滤至 500ml。 该超滤浓缩过程循环 3次。
2、 向 500ml超滤浓缩液中加入 1500ml 0.01M pH7.4 PBS， 按 35 - 40ml/min流速上样至 DEAE - Sephrose FF柱。
3、上样后，依次分别用 0.02M、0.06M和 lM NaCl的 0.01M pH7.4 PBS进行洗脱， 洗脱速度 70-80ml/min。
4、 目的蛋白出现在 0.06M NaCl溶液洗脱液中， 洗脱峰共收集 6750ml。
5、将收集液采用 Millipore Pellicon 2 切向流超滤器（ 0.1M2cut off 8k膜）超滤浓缩至 500ml。
6、 向 500ml超滤浓缩¾中加入 1500ml 0.01M pH7.4的 PBS， 再 超滤至 500ml。 该超滤浓缩过程循环 3次。
7、 向 500ml超滤浓缩液中加入 1500ml 0.01M pH7.4的 PBS, 按 35 - 40ml/min流速上样至 DEAE - Sephrose FF柱。
8、 上样后， 依次分别用 0.02M、 0.04M、 0.06M和 1M NaCl的 0.01M pH7.4 PBS各 10升进行洗脱， 洗脱速度 70-80ml/min。
9、经酶活测定确认目的蛋白出现在 0.06M NaCl溶液洗脱液的第 二个洗脱峰中， 该洗脱峰共收集 3050ml。 PAGE检查为一条带， 说明 已经得到纯化。
10、 将 3050ml 收集液釆用 Millipore Pellicon 2 切向流超滤器 ( 0.1M2cut off 8k膜）进行超滤浓缩至 300ml。 将该 300ml浓缩液上 样至 Sephadex-G25柱上， 用无离子水洗柱脱盐， 收集洗脱峰 490ml。 测定该溶液中总蛋白为 752mg, 比活力为 183u/mg蛋白， 最终收率为 0.75%。 HPLC纯度达 98.0 %， 色谱图与实施例 1相一致。
11、 按滤夜体积加入 1%的低分子右旋糖苷 20冻干保护剂后用 Millipore Viresolve NFP Filters OptiScale - 25 病毒膜过滤。
12、 滤液直接分装后进行冷冻干燥。
13、冻干粉经 PAGE检查为一条带，还原 SDS - PAGE为两条带， 其分子量分别大致为 15kD和 14.5kD。 实施例 4 尖吻蝮蛇血凝酶的丝氨酸蛋白属性实验
将实施例 2分离得到的血凝酶再经 HPLC纯化制备，获得经三维 HPLC 测定蛋白纯度为 100 %的尖吻蝮蛇血凝酶溶液， 酶活性为 10u/mL
用生理盐水配制 1 %的牛纤维蛋白原 （Sigma公司）溶液。
用异丙醇溶解苯甲基磺酰氟（PMSF, Merck公司）， 溶液浓度为 4mg/ml。
实验搡作步骤如下：
( 1 ) 取 1%牛纤维蛋白原溶液 2ml, 37°C下恒温 5分钟。
( 2 ) 取三支小试管， 分别标注 1#、 2#、 3#, 每管加入 200μ1经蒸馏 水稀释一倍的血凝酶溶液 （5u/ml )。
( 3 ) 分别向 1#试管加入 ΙΟμΙ蒸馏水， 2#试管加入 ΙΟμΙ异丙醇， 3# 试管加入 lO l PMSF, 于 37°C水洛保温 5分钟。
( 4 ) 按试管编号顺序分别单独进行凝集试验观察。 向试管中加入恒 温好的 1 %牛纤维蛋白原溶液 200μ1， 立即计时， 同时轻轻摇动 混匀， 于 37°C水洛中静置， 观察试管中凝集反应的情况。 以溶 液呈现完全凝固状态时终止计时。
结果见表一
PMSF对凝集时间的影响
Figure imgf000009_0001
溶液 5u/ml
1# 200μ1 200μ1 ΙΟμΙ蒸馏水 50秒
2# 200μ1 200μ1 ΙΟμΙ异丙醇 50秒
3# 200μ1 200μ1 ΙΟμΙ PMSF 大于 600秒 根据表一中的实验结果，得出以下结论：① lOOppm浓度的 PMSF 完全抑制了该血凝酶活性， 证明该尖吻蝮蛇血凝酶为丝氨酸蛋白酶。 ②微量异丙醇对本凝集反应无影响。 实施例 5 尖吻蝮蛇血凝酶水解人纤维蛋白原实验
用 50mM Tris-HCl(pH7.4)缓冲液配制 4mg/ml的人纤维蛋白原溶 液。 分别向 5支小试管中各加入 0.5ml人纤维蛋白原溶液， 分别按 4 个不同水解保温时间顺序间隔向其中 4管中加入经提取纯化的 1个活 性单位的血凝酶。 37°C水洛保温 4、 2、 1和 0.5小时， 保温后立即进 行 SDS-PAGE 电泳， 观察人纤维蛋白原的水解程度。 另一支 0.5ml 纤维蛋白原对照管同时于 37 °C水浴保温 4小时。
如图 1结果所示： 未经血凝酶水解的纤维蛋白原对照的电泳图呈 现出三条带， 图中 F列从上至下分别为 01、 β、 γ亚基。 纤维蛋白原在 血凝酶（1单位）作用 0.5小时和 1小时后， α亚基带逐渐变浅， 作 用于 2小时和 4小时后， α亚基带完全消失。 该结果证实血凝酶有水 解纤维蛋白原 α亚基的作用。 附 录
尖吻蝮蛇血凝酶单位定义及活性测定方法
①牛纤维蛋白原测定法 取生理盐水配制的 1.0 %牛纤维蛋白原 （Sigma公司） 溶液 lml置小试管中， 37±0.5°C水浴保温 3分钟， 加入 37±0.5°C预热的待测酶溶 液 lml, 立即计时， 纤维蛋白原溶液在 120士30秒内振摇出现白色絮团， 则该酶 溶液为 lu/ml。
②标准人血浆测定法 取标准人血浆 1ml置小试管中，置 37°C±0.5°C水浴中预热 3分钟， 加入 37±0.5°C预热的待测酶溶液 lml， 立即计时， 人血浆在 60±20秒内 振摇出现白色絮团， 则该酶溶液为 lu/ml。
注：测定未知高酶活性溶液时需用无离子水进行稀释，直至达到 lu/ml用于测定; 其稀释倍数即为每毫升原酶溶液中的酶活单位数。

Claims

权 利 要 求 书
1、 尖吻蝮蛇血凝酶， 该酶由 α、 β两个亚基构成， 亚基链间由七 个二硫键连接， 其中 α亚基含 .129个氨基酸， 具有 SEQIDNo.l所示 的氨基酸序列； β亚基含 123个氨基酸， 具有 SEQIDNo.2所示的氨 基酸序列。
2、 如权利要求 1所述的尖吻蝮蛇血凝酶， 其特征在于该酶是一 种丝氨酸蛋白酶， 能够水解人纤维蛋白原 α链。
3、 含有权利要求 1或 2所述尖吻蝮蛇血凝酶的药物。
4、 如权利要求 3所述的药物， 其为冻干粉、 止血贴剂或液体喷 雾剂。
5、 权利要求 1或 2所述血凝酶的纯化方法， 其包括如下步骤：
1)、 蛇毒预处理；
2)、将预处理后的蛇毒溶液上经预平衡的 DEAE - Sepharose Fast Flow阴离子交换层析柱，用 0.01M pH7.0 ~ 7.5 PBS洗柱，再用含 0~1M NaCl的 0.01MpH7.0~7.5的 PBS分段洗脱， 收集 0.06M NaCr溶液 的洗脱峰；
3)、 将上述洗脱液适当浓缩后透析或经反复稀释超滤浓缩去除 NaCl;
4)、 将透析后的溶液再次上经预平衡的 DEAE - Sepharose Fast Flow层析柱， 用 0.01MpH7.0~7.5的 PBS洗柱， 再用含 0~lMNaCl 的 0.01MpH7.0~7.5的 PBS分段洗脱， 收集 0.06M NaCl溶液的第二 个洗脱峰；
5 )、 将上述收集洗脱液适当浓缩后用无离子水透析或经 Sephdex-G25柱去除 NaCl。
6、 如权利要求 5所述的方法， 其中， 步骤 1)蛇毒预处理的方 法是将蛇毒用适量预冷的 0.01M pH7.0~7.5的 PBS溶解， 离心取上 清液进行透析或经反复稀释超滤浓缩。
7、 如权利要求 6所述的方法， 其特征在于其中步骤 1 )蛇毒 1 处理的方法是： 称取蛇毒若干克， 用蛇毒重量 5~10 倍体积预冷的 0.01M pH7.0 ~ 7.5的 PBS于 4〜8°C的层析柜中搅拌溶解 30〜60分钟， 于 4~8°C、5,000~10，000g离心 10〜20分钟，将离心上清液倾入透析袋， 离心沉淀再用蛇毒重量 5~10倍体积预冷的 PBS搅拌悬浮，再次离心， 合并两次离心上清液于透析袋中，在层析柜中 4~8°C对 0.01M pH7.0 ~ 7.5的 PBS透析 12〜24小时，期间更换溶液 2〜4次；或将蛇毒用 10~20 倍体积预冷的 0.01M pH7.0 - 7.5 的 PBS 溶解后， 用分子截留值为 5,000~10，000D的超滤膜经反复多次加入 PBS进行稀释超滤浓缩。
8、 如权利要求 5所述的方法， 其中， 步骤 2 ) 和步骤 4 )釆用 0.01M pH7.0 ~ 7.5 的 PBS预平衡 DEAE - Sepharose Fast Flow层析 柱， 然后上样。
9、 如权利要求 5所述的方法， 其还包括将步骤 5 ) 除盐后的溶 液直接冷冻干燥， 或加入保护剂冷冻干燥。
10、如权利要求 5~9任一项所述的方法， 其特征在于釆用分子截 留值为 5，000〜10，000D超滤膜的超滤浓缩方式降低目的蛋白洗脱溶液 体积， 然后进行透析脱盐； 或釆用分子截留值为 5,000~10，000D的超 滤膜将洗脱液经反复稀释超滤脱盐并浓缩蛋白。
PCT/CN2009/000230 2008-09-27 2009-03-04 来自尖吻蝮蛇的类凝血酶 Ceased WO2010034176A1 (zh)

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