WO2008020743A2 - Method for extracting a platelet aggregation inhibitor from agkistrodon blomhoffii ussuriensis venom - Google Patents

Method for extracting a platelet aggregation inhibitor from agkistrodon blomhoffii ussuriensis venom Download PDF

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Publication number
WO2008020743A2
WO2008020743A2 PCT/MN2006/000005 MN2006000005W WO2008020743A2 WO 2008020743 A2 WO2008020743 A2 WO 2008020743A2 MN 2006000005 W MN2006000005 W MN 2006000005W WO 2008020743 A2 WO2008020743 A2 WO 2008020743A2
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buffer
tris hcl
column
poison
hcl buffer
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PCT/MN2006/000005
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French (fr)
Russian (ru)
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WO2008020743A3 (en
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Georgiy Volkov
Alexiy Savchuk
Vitaly Karbovskyy
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Wisdom Asset Company
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • 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

Definitions

  • the invention relates to the industry of bioactive substances and can be used in biotechnological industries, medicine and the pharmaceutical industry, namely the production of medicines and diagnostic preparations from snake venom.
  • a platelet aggregation inhibitor is a protein that has an inhibitory effect on the platelet activation process by various activation agents.
  • platelet activation inhibitors There are a significant number of platelet activation inhibitors from various sources. All of them differ in the strength of inhibition and the mechanisms by which this inhibition is carried out.
  • One of the effective sources of platelet activation inhibitors is snake venoms, which contain only protein components and have a stable composition.
  • Some methods for producing active enzymes from snake venom are known.
  • a method is known for producing active enzymes from the snake venom of Triteresir okppaepsis by fractionation of poisoned with buffer on Sephadex G-100, chromatography on SM-Touorerl 650M in two stages and final purification on Mono Q gel.
  • Purificapofepofitofibf -Race spake (Agistrodop asites) venom., Toxicon.-1999.-V.37, N7.-P.999-1013); from Yard Agcistrodop Asitis, in which the poison dissolved in the buffer is subjected to separation of the DEAE Sephadex A-50 anion exchanger, then chromatographed twice on Sephadex G-200 (Quaupg C, Hong J. et al. Aspirates Vpom apd its comraritiop with bovipe tlombip., Thromb.Diath.haymorrth. -1971.
  • a method for isolating snake venom of platelet aggregation PAl is proposed, the essence of which includes a method for determining the ability to inhibit platelet aggregation in snake venom, a method for purifying an inhibitor of platelet activity from various samples of snake venom, its characterization and a truncated form containing a modified lysine residue that specifically inhibits fibrin binding or von Willebrand factor with GPPb-Sha. (WO 90/15620, 06/16/1989, Platellet agregatiphibitors, COR ⁇ S, INC).
  • the disadvantage of the prototype is both a low level of profitability of the production process, a low level of purity and a large investment of time.
  • the basis of the invention is the task to develop a method for isolating a platelet activation inhibitor from Agkistrodop Yothqffii poison syriepsis poison, which allows by means of affinity chromatography followed by ion exchange chromatography and the use of carriers with higher chromatographic properties and physicochemical stability to increase the profitability of the production process and reduce the isolation time / fibrinoliptic protein and increase its purity.
  • the problem is solved in a method of an inhibitor of platelet activation from the Agkistrodop Yothoffii venereus poison in that affinity chromatography is carried out on a column with a carrier of Clin Serharose FF using 1OmM Tris HCl with pH 7.4 as a buffer solution.
  • the fraction of a material unrelated to Blue Serum FF was diluted 8 times with 5OmM Tris HCl buffer with pH 8.2 and applied to a column filled with DEAE Serophoros FF anion exchanger, previously equilibrated with 5OmM Tris HCl buffer with pH 8.2.
  • the platelet activation inhibitor was eluted with 5OmM Tris HCl buffer at pH 8.2 with a content of 0.2M NaCl.
  • the protein peak containing the platelet activation inhibitor is collected, concentrated, and applied to replace the buffer with a Serhadex G25 column equilibrated with 5OmM Tris HCl buffer with a pH of 7.4 containing 0.1 ZM NaCl.
  • the problem is solved by the proposed method, when the parameters of the process of isolation of platelet activation inhibitorg are in the ranges indicated in the claims. When the parameters are changed in the direction of increasing or decreasing, the characteristics of the resulting preparation worsen.
  • the solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (60 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • a protein peak containing a platelet activation inhibitor is collected (6 ml) and dried by lyophilization. Characteristic Yield, mg 2.0 mg (4.2%)
  • the solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min )
  • 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (53 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength of 0.2 M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the protein peak containing the platelet activation inhibitor is collected (5.2 ml) and dried by lyophilization. Characteristic Yield, mg 1.7 mg (3.9%) Biological activity IC 5O 232nM
  • the solution of the poison is applied to a column (CU / 10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate of 0.5 ml / min).
  • 1OmM Tris HCl buffer with pH 7.4 is passed through the column at a speed of 2 ml / min and all material unbound with the sorbent (75 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • a protein peak containing a platelet activation inhibitor is collected (7.1 ml) and dried by lyophilization.

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Abstract

The invention relates to producing bioactive substances and can be used for biotechnological production processes, in medicine and in the pharmacological industry, in particular to producing medicinal and diagnosis preparations from snake venom. The inventive method consists in carrying affinity chromatography on a column with a Blue Sepharose FF carrier using a Tris HCl with a PH of 7.4 in the form of a buffer solution, in diluting 8 times a material fraction unconjugated with Blue Sepharose in the Tris HCl buffer with a pH of 8.2 and in applying it to a column which is filled with a DEAE Sepharose FF anion exchanger and is pre-balanced by said Tris HCl buffer, in eluting a platelet aggregation inhibitor in the Tris HCl buffer containing NaCl, in collecting and concentrating the protein peak containing the platelet aggregation inhibitor and, with a view to substitute a buffer, in applying said peak to a column with Sephadex G25, which is balanced with the NaCl-containing HCl buffer, and in drying it by lyophilising.

Description

Название изобретения: Title of invention:
Способ выделения ингибитора агрегации тромбоцитов из яда Аgкistrоdоп Ыотhоffii иssиriепsis I Меthоd fоr рrоduсtiоп оf thrоmbосуtе аggrеgаtiоп iпhibitоr trоmbосitе frоm vепоm Аgкistrоdоп Ыотhоffii иssиriепsisThe method of isolation of platelet aggregation inhibitor from the poison Agcistrodeop Yottoffii Issyriepsis I Metod forporodustiop of thrombosut aggregipotibotor trumpotropod
МПК: А 61 К 35/588 IPC: A 61 K 35/58 8
Область техники к которой относится изобретенияFIELD OF THE INVENTION
Изобретение относится к промышленности биоактивных веществ и может быть использовано в биотехнологических производствах, медицине и фармацевтической промышленности, а именно к производству лекарственных и диагностических препаратов из яда змей.The invention relates to the industry of bioactive substances and can be used in biotechnological industries, medicine and the pharmaceutical industry, namely the production of medicines and diagnostic preparations from snake venom.
Уровень техникиState of the art
Ингибитор агрегации тромбоцитов-белок, обладающей инrибиторным действием на процесс активации тромбоцитами различными агентами активации.A platelet aggregation inhibitor is a protein that has an inhibitory effect on the platelet activation process by various activation agents.
Существует значительное количество ингибиторов активации тромбоцитов из различных источников. Все они отличаются по силе ингибирования и механизмам, по которым осуществляется данное ингибирование. Одним из эффективных источников ингибиторов активации тромбоцитов являются змеиные яды, которые содержат только белковые компоненты и имеют стабильный состав.There are a significant number of platelet activation inhibitors from various sources. All of them differ in the strength of inhibition and the mechanisms by which this inhibition is carried out. One of the effective sources of platelet activation inhibitors is snake venoms, which contain only protein components and have a stable composition.
Известены некоторые способы получения активных ферментов из яда змеи. Например, известен способ получения активных ферментов из яда змеи Тriтеrеsиrиs окгпаvепsis путем фракционирования растворенного буфером яда на Сефадексе G-100, хроматографии на СМ-Тоуореаrl 650M в два этапа и заключительной очистки на Mono Q геле. (Nоsе S., Shimоhigаshi Y. еt аl. Рurifiсаtiоп апd сhаrасtеrizаtiоп оf а соаgulапt епzуmе, оkiпахоbiп II, frоm Тriтеrеsиrиs оkiпаvепsis (Нiтеhаbи sпакё) vепоm whiсh rеlеаsеs fibrinopeptides A апd В., Toxicoп.,-1994.-V.32,-P.1509-1520); из сырого яда змей рода Аgкistrоdоп sахаtilis, в котором сырый яд, растворенный в буфере, подвергается поэтапному фракцированию на Q-сефарозе, Сефадексе G-75 и Mono Q геле (Коh.Y., Сbдmg К., Кim.D. Вiосhеmiсаl сhаrасtеrizаtiоп оf а thrоmbiп-likе епzуmе апd а fibriпоlуtiс serine protease from snake (Аgкistrоdоп sахаtillis) vепоm., Тохiсоп. -2001. — V.39, N4. -P.555-560); из яда Аgкistrоdоп асиtиs, в котором, растворенный в буфере яд подвергают хроматографии на DЕАЕ-Сефарозе, Сефакриле S-200 и СМ-Сефарозе (Нuапg Q., Teng M., Niu L.Рurifiсаtiоп апd сhаrасtеrizаtiоп оf twо fibriпоgеп-сlоttiпg епzуmеs frоm fivе-расе sпаkе {Аgкistrоdоп асиtиs) venom.,Toxicon.-1999.-V.37, N7.-P.999-1013); из ярд Аgкistrоdоп асиtиs, в котором, растворенный в буфере яд подвергают разделению анионообменнике DEAE Сефадексе A-50, затем дважды хроматографируют на Сефадексе G-200 (Quуапg С, Hong J. еt аl. Рurifiсаtiоп апd рrореrtiеs оf thе trоmbiп-likе рriпсiрlе оf Аgкistrоdоп асиtиs vепоm апd its соmраritiоп with bоviпе tlтrоmbiп., Тhrоmb.Diаth.hаеmоrrth. -1971. -V.26 -P.224-234); из сырого яда змей рода Аgкistrоdоп асиtиs, в котором растворенный в буфере (рН 7.2), яд подвергают последовательной хроматографии на Сефадексе G-75, анионообменнике DЕАЕ-Сефадексе A-50 с градиентомд хлорида натрия 0.01-0.6M, сорбенте Гепарин-Сефарозе CL-6B (градиент хлорида натрия NaCl 0.1-0.3; 0.5M) и в отдельном случае дополнительно на Сефадексе G-100. (Коmоri Y, Nikаi Т.еt аl. А соmраrаtivе studу оf сlоttiпg fасtоrs frоm thе vепоm оf Аgкistrоdоп асиtиs соllесtеd iп сhiпа апd Таiwап., Соmр.Вiосhеm.Рhуsiоl. -1987. -V.88B, N2. -P643-649); из сырого яда змей Воthrорs аtrох, заключающийся в выделении тромбиноподобной фракции, способной вызывать коагуляцию фибриногена. (Ноllеmап W.H., Wеiss LJ., thе thrоmbiп-likе епzуmе frоm Воthrорs аtrох аmiпоbепzаmidiпе-substitutеd аgаrоsе. J Вiоl.Сhеm. 1976, Маг. -25. -251(6). —Р.1663-1669); кроме того известен способ, храктеризующий тем, что растворенный в буферном растворе яд подвергают поэтапной хроматографии на Сефадексе G-100, на катионообменнике СМ-Тоуореаrl 650M с линейным градиентом концентрации хлорида натрия и полным объёмом градиента, обеспечивающими наиболее полное выявление и раздельное элюирование тромбиноподобных ферментов, затем проводят раздельную рехроматографию этих ферментов, задавая линейные градиенты концентрации хлорида натрия из условий элюирования соответствующего тромбоподобного фермента на предыдущем этапе при одновременном увеличении полных объемов этих градиентов до уровня, обеспечивающего дополнительное разделение соседных тромбиноподобных ферментов и получают целевой продукт в гомогенном состоянии после раздельной очистки тромбиноподобных ферментов на сорбенте ABA-TSK. (RU 2271219 Cl (2004123274/15, 28.07.2004) 10.03.2006 Бюл.7., Способ получения тромбиноподобных ферментов из яда змеи).Some methods for producing active enzymes from snake venom are known. For example, a method is known for producing active enzymes from the snake venom of Triteresir okppaepsis by fractionation of poisoned with buffer on Sephadex G-100, chromatography on SM-Touorerl 650M in two stages and final purification on Mono Q gel. (Nose S., Shimohigashi Y. et al. Rurifiсtiop apd сhаrаsterizatiop оf а coаgulapt еpzume, okіpakhоbіp II, frоtеrеsіrіs іkіpеpеpісепіпепепепепепіпепіпепепіпепіпепіпепіпепіпепіпепіпепепіпепіпепепіпепепіпепіпепіпепіпепіпепіпепепіпепіпепепепепіпепіпепіпепепіпепепіпепепіпепеепе P.1509-1520); from raw poison of snakes of the genus Agistrodop sakhatilis, in which raw poison, dissolved in a buffer, is subjected to gradual fractionation on Q-Sepharose, Sephadex G-75 and Mono Q gel (Koh.Y., Cbdmg K., Kim.D. and thrombip-likе epzum apd a fibriolutis serine protease from snake (Agistrodop sakhatillis) Vepom. -2001. - V.39, N4. -P.555-560); from Agistrodop asiitis poison, in which the poison dissolved in the buffer is subjected to chromatography on DEAE-Sepharose, Sephacryl S-200, and CM-Sepharose (Huapg Q., Teng M., Niu L. Purificapofepofitofibf -Race spake (Agistrodop asites) venom., Toxicon.-1999.-V.37, N7.-P.999-1013); from Yard Agcistrodop Asitis, in which the poison dissolved in the buffer is subjected to separation of the DEAE Sephadex A-50 anion exchanger, then chromatographed twice on Sephadex G-200 (Quaupg C, Hong J. et al. Aspirates Vpom apd its comraritiop with bovipe tlombip., Thromb.Diath.haymorrth. -1971. -V.26 -P.224-234); from crude venom of the genus Agistrodop asites, in which the poison, dissolved in a buffer (pH 7.2), is subjected to sequential chromatography on Sephadex G-75, an anion exchanger DEAE-Sephadex A-50 with a gradient of sodium chloride 0.01-0.6M, sorbent Heparin-Sepharose CL 6B (gradient of sodium chloride NaCl 0.1-0.3; 0.5M) and, in a separate case, additionally on Sephadex G-100. (Komori Y, Nikita T. et al. And a complimentary stud of сlоttiпg fоtors frоm thе vеpоm оf Аgkistrodop аsіtіs сoіlеstіd іp сіпіп адd Taiwap., Comp. Вісхеm.-8. ; from the raw poison of snakes Віthrоs аtroh, which consists in the isolation of a thrombin-like fraction that can cause coagulation of fibrinogen. (Butlemap WH, Weiss LJ., Thеrombi-likе аbout frоm thrоtors аtroh аmіpobepzamidіpe-substitutеd аgarosе. J Віl.Сhem. 1976, Mag. -25. -251 (666-166-166-166-166). in addition, a method is known that characterizes that the poison dissolved in the buffer solution is subjected to stepwise chromatography on Sephadex G-100, on a SM-Touorerl 650M cation exchanger with a linear gradient of sodium chloride concentration and a full gradient volume, which provide the most complete identification and separate elution of thrombin-like enzymes, then separate rechromatography of these enzymes is carried out, setting linear gradients of the concentration of sodium chloride from the elution conditions of the corresponding thrombus-like enzyme to at the previous stage, while the total volumes of these gradients were increased to a level that provides additional separation of neighboring thrombin-like enzymes, the target product is obtained in a homogeneous state after separate purification of thrombin-like enzymes on an ABA-TSK sorbent. (RU 2271219 Cl (2004123274/15, 07.28.2004) 10.03.2006 Bul. 7., A method of obtaining thrombin-like enzymes from snake venom).
Кроме того предложен способ выделения змеиного яда агрегации тромбоцитов PAl сущность которого включает способ определения в змеином яде способности ингибировать агрегацию тромбоцитов, способ очистки ингибитора активности тромбоцитов из различных образцов змеиного яда, его храктеристику и усеченную форму, содержащую модифицированный остаток лизина, которая специфически ингибирует связывание фибриногена или фактора фон Виллебранда с GPПb-Шa.(WO 90/15620, 16.06.1989, Рlаtеlеt аggrеgаtiоп iпhibitоrs, COR ТНЕRАРЕUТIСS, INC).In addition, a method for isolating snake venom of platelet aggregation PAl is proposed, the essence of which includes a method for determining the ability to inhibit platelet aggregation in snake venom, a method for purifying an inhibitor of platelet activity from various samples of snake venom, its characterization and a truncated form containing a modified lysine residue that specifically inhibits fibrin binding or von Willebrand factor with GPPb-Sha. (WO 90/15620, 06/16/1989, Platellet agregatiphibitors, COR ТНЕРАРУТИСS, INC).
Недостатком прототипа является как низкий уровень рентабельности производственного процесса, низкий уровень чистоты и большой затрат времени.The disadvantage of the prototype is both a low level of profitability of the production process, a low level of purity and a large investment of time.
Сущность изобретенияSUMMARY OF THE INVENTION
В основу изобретения поставлена задача разработать способ выделения ингибитора активации тромбоцитов из яда Аgkistrоdоп Ыотhqffii иssиriепsis, позволяющий путем проведения афинной хроматографии с последующей ионообменной хроматографией и применения носителей, обладающих более высокими хроматографическими свойствами и физико-химической стабилностью, повысить рентабельность производственного процесса, сократить время выделения фибриногено/фибринолиптического белка и повысить его чистоту. Поставленная задача решается в способе ингибитора активации тромбоцитов из яда Аgkistrоdоп Ыотhоffii иssиriепsis в том, что аффинную хроматографию проводят на колонке с носителем Вlие Sерhаrоsе FF, используя в качестве буферного раствора 1OmM Тris HCl с рН 7.4. ,Фpaкцию несвязанного с Вlие Sерhаrоsе FF, материала разбавляют 8 раз 5OmM Тris HCl буфером с рН 8.2 и наносят на колонку заполненную анионообменником DEAE Sерhаrоsе FF, предварительно уравновешенную 5OmM Тris HCl буфером с рН 8.2. После отмывания сорбента от несвязанного материала, ингибитор активации тромбоцитов элюируют 5OmM Тris HCl буфере с рН 8.2 с содержанием 0.2M NaCl. Белковый пик, содержащий ингибитор активации тромбоцитов, собирают, концентрируют и с целью смены буфера наносят на колонку с Sерhаdех G25, уравновешенную 5OmM Тris HCl буфером с рН 7.4 с содержанием 0.1 ЗМ NaCl. Осуществление изобретенияThe basis of the invention is the task to develop a method for isolating a platelet activation inhibitor from Agkistrodop Yothqffii poison syriepsis poison, which allows by means of affinity chromatography followed by ion exchange chromatography and the use of carriers with higher chromatographic properties and physicochemical stability to increase the profitability of the production process and reduce the isolation time / fibrinoliptic protein and increase its purity. The problem is solved in a method of an inhibitor of platelet activation from the Agkistrodop Yothoffii venereus poison in that affinity chromatography is carried out on a column with a carrier of Clin Serharose FF using 1OmM Tris HCl with pH 7.4 as a buffer solution. , The fraction of a material unrelated to Blue Serum FF was diluted 8 times with 5OmM Tris HCl buffer with pH 8.2 and applied to a column filled with DEAE Serophoros FF anion exchanger, previously equilibrated with 5OmM Tris HCl buffer with pH 8.2. After washing the sorbent from unbound material, the platelet activation inhibitor was eluted with 5OmM Tris HCl buffer at pH 8.2 with a content of 0.2M NaCl. The protein peak containing the platelet activation inhibitor is collected, concentrated, and applied to replace the buffer with a Serhadex G25 column equilibrated with 5OmM Tris HCl buffer with a pH of 7.4 containing 0.1 ZM NaCl. The implementation of the invention
Поставленная задача решается предлагаемым способом, когда параметры процесса выделения ингибиторg активации тромбоцитов находятся в переделах, указанных в формуле изобретения. При изменении параметров в сторону увеличения или уменьшения характеристики получаемого препарата ухудшаются.The problem is solved by the proposed method, when the parameters of the process of isolation of platelet activation inhibitorg are in the ranges indicated in the claims. When the parameters are changed in the direction of increasing or decreasing, the characteristics of the resulting preparation worsen.
Изобретение иллюстрируется примерами 1-3 конкретного осуществления.The invention is illustrated by examples 1-3 of a specific implementation.
Пример.1Example 1
К 100мг яда щитомордника дальневосточного добавляют 5мл 1OmM Тris HCl буфера с рН5 ml of 1OmM Tris HCl buffer with pH is added to 100 mg of Far Eastern muzzle poison
7,4 и перемешивают до полного растворения яда при 40C. Растворенный яд центрифугируют при 5000 об/мин при 40C в течении 15 мин. Осадок отбрасывают а надосадочную жидкость используют для афинной хроматографии. Раствор яда наносят на колонку (С 10/10, объём 7мл) с афинным сорбентом Вlие Sерhаrоsе FF, предварительно уравновешенную 50мл 1OmM Тris HCl буфером с рН 7,4 (скорость уравновешивания колонки 2 мл/мин, скорость нанесения яда 0,5мл/мин). После нанесения, через колонку со скоростью 2мл/мин пропускают 1OmM Тris HCl буфера с рН 7,4 и собирают весь несвязанный с сорбентом материал (60мл). Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Фракцию несвязанного с Вlие Sерhаrоsе FF материала разбавляют в 8 раз 5OmM Тris HCl буфером с рН 8.2 и наносят на колонку заполненную анионообменником DEAE Sерhаrоsе FF (размер колонки Ix8cм, объем 7мл), предварительно уравновешенную 5OmM Тris HCl буфером с рН 8.2 со скоростью 2 мл/мин (Скорость нанесения образца lмл/мин). Основная часть белков (85%) собируется на анионообменнике. После отмывания сорбента от несвязанного материала буфером нанесения (120мл), ингибитор активации тромбоцитов элюируют ступенчатым градиентом ионной силы 0.2M NaCl в 5OmM Тris HCl буфере с рН 8.2 со скоростью 2мл/мин. Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Белковый пик содержащий ингибитор активации тромбоцитов собирают (6мл) и высушивают лиофилизацией. Характеристика Выход, мг 2.0 мг (4.2%)7.4 and stirred until the poison is completely dissolved at 4 0 C. The dissolved poison is centrifuged at 5000 rpm at 4 0 C for 15 minutes. The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (60 ml) is collected. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger. After washing the sorbent from the unbound material with the application buffer (120 ml), the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). A protein peak containing a platelet activation inhibitor is collected (6 ml) and dried by lyophilization. Characteristic Yield, mg 2.0 mg (4.2%)
Биологическая активность IC50 23OnM Пример 2.Biological Activity IC 50 23OnM Example 2
К 96мг яда щитомордника дальневосточного добавляют 5мл 1OmM Тris HCl буфера с рН 7,4 и перемешивают до полного растворения яда при 40C. Растворенный яд центрифугируют при 5000 об/мин при 40C в течении 15 'мин. Осадок отбрасывают а надосадочную жидкость используют для афинной хроматографии. Раствор яда наносят на колонку (С 10/10, объём 7мл) с афинным сорбентом Вlие Sерhаrоsе FF, предварительно уравновешенную 50мл 1OmM Тris HCl буфером с рН 7,4 (скорость уравновешивания колонки 2 мл/мин, скорость нанесения яда 0,5мл/мин). После нанесения, через колонку со скоростью 2мл/мин пропускают 1OmM Тris HCl буфера с рН 7,4 и собирают весь несвязанный с сорбентом материал (53мл). Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Фракцию несвязанного с Вlие Sерhаrоsе FF материала разбавляют в 8 раз 5OmM Тris HCl буфером с рН 8.2 и наносят на колонку заполненную анионообменником DEAE Sерhаrоsе FF (размер колонки Ix8cм, объем 7мл), предварительно уравновешенную 5OmM Тris HCl буфером с рН 8.2 со скоростью 2 мл/мин (Скорость нанесения образца lмл/мин). Основная часть белков (85%) собируется на анионообменнике. После отмывания сорбента от несвязанного материала буфером нанесения (112мл), ингибитор активации тромбоцитов элюируют ступенчатым градиентом ионной силы 0.2M NaCl в 5OmM Тris HCl буфере с рН 8.2 со скоростью 2мл/мин. Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Белковый пик содержащий ингибитор активации тромбоцитов собирают (5.2мл) и высушивают лиофилизацией. Характеристика Выход, мг 1.7 мг (3.9%) Биологическая активность IC5O 232nM5 ml of 1OmM Tris HCl buffer with pH 7.4 is added to 96 mg of Far Eastern thyroid muzzle venom and mixed until the venom is completely dissolved at 4 ° C. The dissolved venom is centrifuged at 5,000 rpm at 4 ° C for 15 min. The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (53 ml) is collected. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger. After washing the sorbent from the unbound material with an application buffer (112 ml), the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength of 0.2 M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The protein peak containing the platelet activation inhibitor is collected (5.2 ml) and dried by lyophilization. Characteristic Yield, mg 1.7 mg (3.9%) Biological activity IC 5O 232nM
Пример 3.Example 3
К 110мг яда щитомордника дальневосточного добавляют 5мл 1OmM Тris HCl буфера с рН 7,4 и перемешивают до полного растворения яда при 40C. Растворенный яд центрифугируют при 5000 об/мин при 40C в течении 15 мин. Осадок отбрасывают а надосадочную жидкость используют для афинной хроматографии. Раствор яда наносят на колонку (СЮ/10, объём 7мл) с афинным сорбентом Вlие Sерhаrоsе FF, предварительно уравновешенную 50мл 1OmM Тris HCl буфером с рН 7,4 (скорость уравновешивания колонки 2 мл/мин, скорость нанесения яда 0,5мл/мин). После нанесения, через колонку со скоростью 2мл/мин пропускают 1OmM Тris HCl буфера с рН 7,4 и собирают весь несвязанный с сорбентом материал (75мл). Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Фракцию несвязанного с Вlие Sерhаrоsе FF материала разбавляют в 8 раз 5OmM Тris HCl буфером с рН 8.2 и наносят на колонку заполненную анионообменником DEAE Sерhаrоsе FF (размер колонки Ix8cм, объем 7мл), предварительно уравновешенную 5OmM Тris HCl буфером с рН 8.2 со скоростью 2 мл/мин (Скорость нанесения образца lмл/мин). Основная часть белков (85%) собируется на анионообменнике. После отмывания сорбента от несвязанного материала буфером нанесения (135мл), ингибитор активации тромбоцитов элюируют ступенчатым градиентом ионной силы 0.2M NaCl в 5OmM Тris HCl буфере с рН 8.2 со скоростью 2мл/мин. Оптическую плотность регистрируют непрерывно с помощью проточного монитора REC 102 ("Раrmасiа biоtесh"). Белковый пик содержащий ингибитор активации тромбоцитов собирают (7.1мл) и высушивают лиофилизацией. Характеристика5 ml of 1OmM Tris HCl buffer with a pH of 7.4 is added to 110 mg of Far Eastern thyroid muzzle venom and mixed until the venom is completely dissolved at 4 ° C. The dissolved venom is centrifuged at 5000 rpm at 4 ° C for 15 minutes. The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (CU / 10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate of 0.5 ml / min). After application, 1OmM Tris HCl buffer with pH 7.4 is passed through the column at a speed of 2 ml / min and all material unbound with the sorbent (75 ml) is collected. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger. After washing the sorbent from the unbound material with the application buffer (135 ml), the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). A protein peak containing a platelet activation inhibitor is collected (7.1 ml) and dried by lyophilization. Characteristic
Выход, мг 2.9 мг (4.85%)Yield, mg 2.9 mg (4.85%)
Биологическая активность IC5O 229nM Biological Activity IC 5O 229nM

Claims

Формула изобретенияClaim
Способ выделения ингибитора активации тромбоцитов из яда Аgкistrоdоп blотhоffii иssиriепsis, включающий растворение яда в буферном растворе с последующим центрифугированием, аффинной хроматографии, и следующей за ней ионнообменной хроматографией, сбор фракций, содержащих биологическую активность ингибитора активации тромбоцитов и лиофилизацию отличающийся тем, что аффинную хроматографию проводят на колонке с носителем Вlие Sерhаrоsе FF, используя в качестве буферного раствора 1OmM Тris HCl с рН 7,4 и затем фракцию несвязанного с Вlие Sерhаrоsе FF материала разбавляют в 8 раз 5OmM Тris HCl буфером с рН 8.2 и наносят на колонку заполненную анионообменником DEAE Sерhаrоsе FF, предварительно уравновешенную 5OmM Тris HCl буфером с рН 8.2; затем отмывают сорбента от несвязанного материала, ингибитор активации тромбоцитов элюируют 5OmM Тris HCl буфере с содержанием 0.02M NaCl, белковый пик содержащий ингибитор активации тромбоцитов собирают, концентрируют и с целью смены буфера наносят на колонку Sерhаdех G25, уравновешенную 5OmM Тris HCl буфером с рН 7.4 с содержанием 0.1 ЗМ NaCl. A method for isolating a platelet activation inhibitor from Agistrodop blothoffii poison syriepsis poison, including dissolving the poison in a buffer solution, followed by centrifugation, affinity chromatography, and subsequent ion exchange chromatography, collecting fractions containing the biological activity of the platelet activating inhibitor and lyophilization, which differs by a column containing Bilie Serfaros FF, using 1OmM Tris HCl pH 7.4 as a buffer solution and then a fraction of unbranched Serfaros FF material diluted 8 times 5OmM Tris HCl buffer pH 8.2 and applied to a column filled with anion exchanger DEAE Serharose FF, pre-equilibrated with 5OmM Tris HCl buffer pH 8.2; then the sorbent is washed from unbound material, the platelet activation inhibitor is eluted with 5OmM Tris HCl buffer containing 0.02M NaCl, the protein peak containing the platelet activation inhibitor is collected, concentrated, and applied to a column of buffer S25 with pH 5 buffer balanced with 5OmM Tris HCl with buffer 0.1 M NaCl.
PCT/MN2006/000005 2006-08-16 2006-08-30 Method for extracting a platelet aggregation inhibitor from agkistrodon blomhoffii ussuriensis venom WO2008020743A2 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1293795A (en) * 1969-07-04 1972-10-25 Twyford Lab Ltd Improvements relating to anticoagulants
WO1990015620A1 (en) * 1989-06-16 1990-12-27 Cor Therapeutics, Inc. Platelet aggregation inhibitors
CN1504569A (en) * 2002-12-05 2004-06-16 合肥兆峰科大药业有限公司 Chromatography purification process of viper venom blood clotting enzyme
RU2271219C1 (en) * 2004-07-28 2006-03-10 Государственное образовательное учреждение высшего профессионального образования "Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова Министерства здравоохранения Российской Федерации" Method for production of thrombin-like enzyme from snake venom

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1293795A (en) * 1969-07-04 1972-10-25 Twyford Lab Ltd Improvements relating to anticoagulants
WO1990015620A1 (en) * 1989-06-16 1990-12-27 Cor Therapeutics, Inc. Platelet aggregation inhibitors
CN1504569A (en) * 2002-12-05 2004-06-16 合肥兆峰科大药业有限公司 Chromatography purification process of viper venom blood clotting enzyme
RU2271219C1 (en) * 2004-07-28 2006-03-10 Государственное образовательное учреждение высшего профессионального образования "Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова Министерства здравоохранения Российской Федерации" Method for production of thrombin-like enzyme from snake venom

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