CN1297896A

CN1297896A - Method of extracting activated prothrombin X(FXa) effectively

Info

Publication number: CN1297896A
Application number: CN 99125207
Authority: CN
Inventors: 吴双顶; 赵超; 王晶莹; 马瑞京; 彭安; 刘清亮
Original assignee: University of Science and Technology of China USTC; Research Center for Eco Environmental Sciences of CAS
Current assignee: University of Science and Technology of China USTC; Research Center for Eco Environmental Sciences of CAS
Priority date: 1999-11-29
Filing date: 1999-11-29
Publication date: 2001-06-06

Abstract

The present invention, the method of extracting activated blood coagulation factor X (FXa) from ox blood, belongs to the field of biological technology. Coarse extraction containing blood coagulation factor X is first extracted from ox blood by modern separation measures, then separated as purified by liquid separation with Q-Sepharose column, and finally activated. By a fast chromatographic liquid phase protein separating process, high purity FXa is obtained. The present invention has the features of less separating steps, short separating period, and high effect and can meet the requirement of production and scientific research.

Description

The method of a kind of high efficiency extraction activatory Stuart factor (FXa)

The invention relates to the problem of utilizing ox blood to make raw material, high efficiency extraction activatory Stuart factor (FXa), belong to biological technical field.

Stuart factor (FX) is a glycoprotein (contains approximately sugar 10%), and it is the thrombin that vitamin K relies on, and in the waterfall reaction of blood coagulation, is positioned at the joint of external source path, so to the process important influence of blood coagulation.The content of Stuart factor in blood plasma it is generally acknowledged about 10 mg/litre.After the reaction of the blood coagulation waterfall in the blood was activated, Stuart factor can normally be activated.Concerning the external source path, activator is the mixture of activatory serum prothrombin conversion accelerator (SPCA) and tissue factor, at Ca ²⁺Carry out under the existence condition.Concerning endogenous path, activator is the activatory plasma thromboplastin component.The activatory platelet cofactor has quickened the latter's reaction greatly as coenzyme.This reaction conditio sune qua non still has Ca ²⁺, also have the existence of phosphatide in addition.Whether and improper activated clotting factor X does not need the participation of coagulation factor protein, have phosphatide to exist and do not require yet, has only required Ca ²⁺Exist and get final product.Vipoxin all over the world generally contains the special lytic enzyme of improper activated clotting factor X, and this special lytic enzyme in the vipoxin has been used in the preparation of activatory Stuart factor (FXa).Some tumour cell also contains the special lytic enzyme of this class in addition.1985 in tumor research; find that malignant cell also can improper activatable Stuart factor in the blood migration; perhaps the localization effect of FXa just; institute is so that tumor cell surface has produced scleroproein; and this blood coagulation layer is as protective shell; defendd entering of natural killer, also can hide immune identification simultaneously, perhaps this be that tumour is striden a kind of mode that tissue migration is achieved.

Anticoagulation and antithrombotic newtype drug are the emphasis of new drug research always.The single-minded better effects if that suppresses FXa than Trombin inhibiting effectively.This is that FXa then stays out of the anticoagulation system of blood because zymoplasm also has important effect in the anticoagulation system of blood.Single-minded effective inhibition FXa can make drug effect improve greatly.Simultaneously, single-minded effective inhibition FXa also may have the effect of antitumor migration.The albumin crystal structure that these researchs all need FXa, particularly modern medicines designing institute to need also will need relatively large FXa.

Because FXa is the very high arginine peptide bond hydrolysis enzyme of selectivity, recently be designed to the limited restriction endonuclease (or claiming specificity lytic enzyme) of the fusion rotein of fusion gene expression by engineered scientist, GST fusion gene carrier as the exploitation of Sweden Pharmacia company, the designed activatory Stuart factor identifier of pGEX series be Ile Glu Gly Arg ↓, compare with other limited restriction endonuclease, its advantage is whole aminoacid sequences of the target protein after can keeping cloning, and can avoid not wishing the interference to the products molecule structure of the amino acid introduced fully.This restriction endonuclease can be with the wish of target protein in the fusion rotein according to the planner, and the restriction enzyme site that designs from fusion rotein cuts down, thereby reached high-purity purpose of separating efficiently.In the genetically engineered in modern times, fusion rotein has been played the part of important role, its characteristic that efficiently expresses, not only come into one's own day by day, particularly it has the character of purifying " label ", has obtained sufficient application in affinity chromatography, also is to bring many facilities on detecting.Limited restriction endonuclease can be used cleverly in conjunction with separation means in fusion rotein separates, and has not only simplified separating step greatly, improve the target protein rate of recovery, and purity generally is better than directly expressing the product of target protein.The production of medicine fusion rotein then is the new focus that activatory thrombin ten is used.

Therefore, efficient production FXa is very significant.

The present domestic report that does not still have this class Separation Research in the existing in the world bibliographical information, all is to be the separation method of purpose with research, and speed is slow, is difficult to satisfy the needs of development in science and technology.For example, the step that contains Stuart factor that people such as Radcliffe utilize the ox blood pre-treatment to obtain is more, utilizes twice separation method of DEAE-Cellulose column chromatography again, and small molecular weight impurity is removed in dialysis again, and disengaging time is longer, and the easy inactivation of FXa.

Purpose of the present invention is utilized modern separation means exactly, by reducing separating step, improves separation efficiency, obtains high purity activatory Stuart factor (FXa) from ox blood, to reach the purpose of widespread use.Enforcement schemes of the present invention: (1) contains the acquisition of the ox blood crude extract of Stuart factor: get 14 liters of ox bloods, in every liter of ox blood, add 100 milliliters of antithrombotics and (contain the 0.2mol/L trisodium citrate, the 17000unit/L heparin, 0.2mol/L benzenyl amidine, the 10000unit/L Trypsin inhibitor SBTI), centrifugal immediately, obtain glassy yellow blood plasma; In every liter of blood plasma, drip 100 milliliters of 1mol/L barium chloride solutions, obtain adsorbing the barium citrate precipitation of Stuart factor; Precipitation is joined the ammoniumsulphate soln of 30% saturation ratio, stirred 3 hours, get clear liquid after centrifugal, make clear liquid ammonium sulfate saturation ratio reach 65% again, left standstill about 5 hours, get the albumen precipitation that contains Stuart factor after centrifugal, dissolving, dialysis, lyophilize, promptly get the ox blood crude extract that contains Stuart factor, for future use.(2) Stuart factor purifying: Q-Sepharose post liquid chromatography (LC)

(1) is handled the freeze dried ox blood crude extract dissolving that 14 liters of ox bloods obtain, and (2.6 * 60cm) liquid chromatography (LC) are carried out albumen sepn to adopt the Q-Sepharose post.Elution speed is 4.0ml/min, and damping fluid is 50mmol/L Tris-HCl (pH8.0) solution, adopts 0-1.0mol/LNaCl straight line gradient elution, and part is collected (8.0ml/ pipe).The 280nm ultraviolet monitoring.Separating resulting such as Fig. 1.Determine to collect at the peak at Stuart factor place with German Bao Ling Man method detection of active, dialysis, freeze-drying, standby.(3) preparation of activatory Stuart factor (FXa)

Activation: the Stuart factor dry powder that will (2) obtains is dissolved into the solution of 20mg/ml, Stuart factor activating enzymes component (RVV-X) and CaCl in every ml soln in the adding Lu Shi vipoxin ₂Solution makes its concentration be respectively 0.5mg/ml and 1mmol/L, and room temperature was placed after 4 hours, separated immediately.

Fast protein liquid chromatography (FPLC): utilize the mixed solution after FPLC (Mono Q post) will activate to carry out separation and purification.Elution speed is 1.0ml/min, and 280nm ultraviolet monitoring, damping fluid are 50mmol/L Tris-HCl (pH8.0) solution, adopts 0-0.6mol/L NaCl straight line gradient elution, presses the peak and collects separating resulting such as Fig. 2.With respectively collection part of German Bao Ling Man's method detection activity, determine the part at FXa place.Adopt traditional polyacrylamide gel electrophoresis method to detect the purity of FXa, it is single pure that detected result such as Fig. 3, FXa reach electrophoresis.

Claims

1, a kind of from ox blood the method for high efficiency extraction activatory Stuart factor (FXa): this method is to extract the crude extract that obtains containing Stuart factor from ox blood, utilize Q-Sepharose post liquid chromatography (LC) to separate and obtain Stuart factor, reactivate it, directly obtain electrophoretically pure FXa then with Mono Q post liquid chromatography (LC) is separable.

2, according to the method for claim 1, in the process that obtains the ox blood crude extract, the saturation ratio that is added in the ammoniumsulphate soln in the barium citrate precipitation is 30%.

3, according to the method for claim 1, the buffering liquid of the used Q-Sepharose post of this method liquid chromatography (LC) is: buffer A: 50mmol/L Tris-HCl, pH8.0; Buffer B: A+1.0mol/L NaCl.And the straight line gradient is 500ml buffer A+500ml buffer B.

4, according to the method for claim l, in the reactivation process of Stuart factor, RVV-X component (RVV-X) add-on in the Lu Shi vipoxin should be 0.1mg/ml, and soak time should be 4 hours.

5, according to the method for claim 1, utilizing FPLC (Mono Q post) when carrying out separation and purification, damping fluid should be 50mmol/L Tris-HCl (pH8.0) solution, adopts NaCl straight line gradient elution, and the NaCl gradient should be 0-0.6mol/L.