阅读说明：本技术 用于纯化组织因子的缓冲组合物、纯化制剂、pt检测组合物以及pt检测试剂盒 (Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit ) 是由 丁重辉 李博华 胡晓娟 于 2019-11-29 设计创作，主要内容包括：本发明涉及检测领域,特别涉及用于纯化组织因子的缓冲组合物、纯化制剂、PT检测组合物以及PT检测试剂盒。本发明所用到的关键原材料组织凝血活酶是通过基因工程的手段制备得到的,通过发酵技术,纯化技术大规模获得,并得到高纯度的r-TF,代替了兔脑,胎盘等组织的来源难,批间差大,敏感性低的问题。本发明基于重组组织因子r-TF液体型的高敏凝血酶原PT诊断试剂盒,稳定性好,批间差小,敏感性高,主要用于临床外源性凝血途径的筛查,口服华法林药物的监控,肝病等疾病的辅助诊断,且可溯源至国际标准品PT试剂上,使得不同参考实验室,不同厂家试剂之间的检测具有可比性。(The invention relates to the field of detection, in particular to a buffer composition, a purification preparation, a PT detection composition and a PT detection kit for purifying tissue factor. The key raw material tissue thromboplastin used by the invention is prepared by means of genetic engineering, is obtained in a large scale by a fermentation technology and a purification technology, and high-purity r-TF is obtained, so that the problems of difficult sources, large batch difference and low sensitivity of rabbit brain, placenta and other tissues are replaced. The high-sensitivity prothrombin PT diagnostic kit based on the recombinant tissue factor r-TF liquid type has the advantages of good stability, small batch difference and high sensitivity, is mainly used for screening clinical extrinsic coagulation paths, monitoring oral warfarin medicines and auxiliary diagnosis of diseases such as liver diseases, and can be traced to an international standard PT reagent, so that the detection of different reference laboratories and reagents of different manufacturers has comparability.)

1. A buffered composition for the purification of tissue factor comprising Triton, Hepes, NaCl and imidazole, said Triton being present at a concentration of 0.5% by volume.

2. The buffered composition of claim 1, comprising an equilibration buffer and an elution buffer; the equilibration buffer comprises the following components:

the elution buffer comprises the following components:

3. use of a buffered composition according to claim 1 or 2 for the preparation of a tissue factor purification formulation.

4. A tissue factor purification preparation comprising a loading buffer and a buffered composition according to claim 1 or 2; the loading buffer comprises the following components:

5. use of the tissue factor purification preparation according to claim 4 for the purification of tissue factor.

A PT assay composition comprising the tissue factor purification preparation of claim 4 and a detection reagent; the detection reagent comprises tissue factor, phospholipid and surfactant.

7. The PT assay composition of claim 6, wherein the tissue factor, the phospholipid and the surfactant are in a mass ratio of 3:10: 30.

8. The PT detection composition of claim 7, wherein the surfactant comprises one or a mixture of Chaps, sodium deoxycholate; the phospholipid comprises one or more of rabbit brain phospholipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine;

the PT assay composition further comprises a heparin antagonist.

9. Use of the PT test composition of any one of claims 6 to 8 in the preparation of a PT test kit.

A PT test kit comprising a PT test composition according to any one of claims 6 to 8 and acceptable adjuvants.

Technical Field

The invention relates to the field of detection, in particular to a buffer composition, a purification preparation, a PT detection composition and a PT detection kit for purifying tissue factor.

Background

The four blood coagulation items belong to one of clinical laboratory examination items, belong to routine screening examination and mainly aim at the detection of extrinsic coagulation factors. The detection principle of PT is as follows: excess tissue thromboplastin and calcium ions are added to the plasma to be tested to convert prothrombin to thrombin, which converts fibrinogen to fibrin. The time for detecting the coagulation of the plasma is the prothrombin time. Prothrombin Time (PT) is one of four routine blood clotting, and PT kits are used for screening of extrinsic coagulation pathway and warfarin monitoring and for auxiliary diagnosis of other diseases.

The most important application is that the International Normalized Ratio (INR) obtained by converting the detection result becomes the first-choice index for monitoring the treatment of the oral anticoagulant drugs. The tissue thromboplastin in PT reagent is generally derived from brain or lung tissue extract of human, bovine rabbit, and monkey. Tissue from different sources has different thromboplastin sensitivity. In 1984, the international committee for standardization in hematology (ICSH) and the International Committee for Thrombosis and Hemostasis (ICTH) proposed labeling thromboplastin with the International Sensitivity Index (ISI), and reporting the PT measurement results using the International Normalized Ratio (INR) reporting method, which is calculated as INR ═ PTR ^ISIWhere PTR is the ratio of PT, i.e. PT (s)/normal human mean mnpt(s) of the patient being examined, ISI is the international sensitivity index, the smaller the international sensitivity index, the higher its sensitivity.

The thromboplastin is derived from the leachate of rabbit brain, placenta and lung tissue. The PT reagent thromboplastin of sysmex is derived from placenta, and the PT thromboplastin of stago, Shanghai Sun, etc. is derived from rabbit brain tissue. Because the thromboplastin sources of different companies are different, the concentrations are different, the sensitivity is different, and the problems of poor product quality, instability, poor sensitivity and the like are caused. The main components of thromboplastin are Tissue Factor (TF) and phospholipids.

Tissue Factor (TF) is a transmembrane single-chain glycoprotein consisting of 263 amino acid residues and having a molecular weight of approximately 47 kDa. The blood coagulation activator mainly plays a role in that the extracellular region contains 3-5 disulfide bond structures, has serine protease activity and is an important part for activating blood coagulation, 23 amino acids immediately after the serine protease activity penetrate through a cell membrane, and the transmembrane region is combined with phospholipid on the surface of the cell membrane and provides a downstream factor including Ca ²⁺And VII (seven factor) bound surfaces. TF is the main active component of the prothrombin time detection reagent and influences thrombinThe main factor of the original time measurement, high quality tissue factor, can sensitively reflect whether the coagulation factor is abnormal or not.

Disclosure of Invention

In view of the above, the present invention provides a buffer composition, a purification preparation, a PT assay composition and a PT assay kit for purifying tissue factor. The invention constructs, expresses and purifies to obtain the active human gene recombinant tissue factor by gene recombination technology, provides a buffer composition for purifying the tissue factor and a purifying reagent to obtain the tissue factor with high purity, and prepares the liquid PT reagent by a lipidation process. The reagent has small difference between batches, high sensitivity, anti-heparin interference, sensitivity to coagulation seven Factor (FVII), and traceability to international standards.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a buffer composition for purifying tissue factor, which comprises Triton, Hepes, NaCl and imidazole, wherein the volume concentration of the Triton is 0.5%.

In some embodiments of the invention, an equilibration buffer and an elution buffer are included; the equilibration buffer comprises the following components:

the elution buffer comprises the following components:

on the basis of the research, the invention also provides application of the buffer composition in preparing a tissue factor purification preparation.

The invention also provides a tissue factor purification preparation, which comprises a loading buffer solution and the buffer composition; the loading buffer comprises the following components:

the invention also provides application of the tissue factor purification preparation in purifying tissue factors.

On the basis of the research, the invention also provides a PT detection composition, which comprises the tissue factor purification preparation and a detection reagent; the detection reagent comprises tissue factor, phospholipid and surfactant.

In some embodiments of the invention, the mass ratio of the tissue factor, the phospholipid and the surfactant is 3:10: 30.

In some embodiments of the invention, the surfactant comprises one or a mixture of Chaps, sodium deoxycholate; the phospholipid comprises one or more of rabbit brain phospholipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine;

the PT assay composition further comprises a heparin antagonist. In some embodiments of the invention, the heparin antagonist comprises one or a mixture of polybrene and protamine.

The invention also provides application of the PT detection composition in preparation of a PT detection kit.

On the basis of the research, the invention also provides a PT detection kit, which comprises the PT detection composition and an acceptable auxiliary agent.

The invention discloses a preparation process of a recombinant factor, a method for constructing a PT kit, and solves the defects caused by the PT kit of rabbit brain tissue. The main raw materials of the prothrombin PT kit used in the market at present are rabbit brain, placenta and other tissues, and the prothrombin PT kit has the defects of low biological activity, poor stability, large batch difference, difficult source and the like, and the preparation technology of the recombinant TF is developed along with the development of the genetic engineering technology. The method aims to prepare the prothrombin liquid type diagnostic reagent which has a low ISI value and high sensitivity compared with a rabbit brain freeze-dried PT reagent by researching the technologies of molecular cloning, fermentation, purification, lipidization and the like. The key raw material tissue thromboplastin used by the invention is prepared by means of genetic engineering, is obtained in a large scale by a fermentation technology and a purification technology, and high-purity r-TF is obtained, so that the problems of difficult sources, large batch difference and low sensitivity of rabbit brain, placenta and other tissues are replaced. The high-sensitivity prothrombin PT diagnostic kit based on the recombinant tissue factor r-TF liquid type has the advantages of good stability, small batch difference and high sensitivity, is mainly used for screening clinical extrinsic coagulation paths, monitoring oral warfarin medicines and auxiliary diagnosis of diseases such as liver diseases, and can be traced to an international standard PT reagent, so that the detection of different reference laboratories and reagents of different manufacturers has comparability.

The beneficial effects of the invention are not limited to: the sensitivity is high (ISI is low) and adjustable, the liquid reagent does not need to be redissolved, the operation steps and the deviation caused by redissolution are reduced, and the anti-heparin effect is obvious.

Detailed Description

The invention discloses a buffer composition, a purification preparation, a PT detection composition and a PT detection kit for purifying tissue factor, and a person skilled in the art can use the contents to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Interpretation of terms:

pt (prothrombin time): i.e. prothrombin time, prothrombin is converted to thrombin by adding tissue thromboplastin and calcium ions in vitro, fibrinogen is converted to fibrin by the generated thrombin, and the time required for the platelet-deficient plasma to coagulate is calculated as prothrombin time PT.

r-TF (recombinant tissue factor): namely, the recombinant tissue factor is also the coagulation factor III, the tissue factor exists in organs such as tissue placenta, brain, heart and lung, is a key substance for activating an extrinsic coagulation pathway, can further activate the coagulation factor VII, is combined with the factor VII to activate the factor X, can also activate the factor IX through a bypass pathway, and then activates an intrinsic coagulation pathway. r-TF is a tissue factor obtained by molecular cloning, protein in vitro expression and purification by using a genetic engineering method.

Isi (international Sensitivity index): i.e. the international sensitivity index, used to calculate the international normalized ratio INR, each reagent manufacturer will give ISI values for different batches on different instruments.

Inr (international Normalized ratio): i.e. the international normalized ratio, INR was calculated using the PT ratio of the reference plasma to normal plasma measured with thromboplastin and the ISI value plotted against the reagent used, so that the results measured with the different thromboplastin reagents were comparable. Calculated by the formula:

extracting RNA (ribonucleic acid) in tissues, designing a primer, carrying out mass amplification through PCR (polymerase chain reaction), introducing into recombinant plasmids, transforming and introducing competence to obtain recombinant strains, fermenting and expressing, crushing bacterial cells, and carrying out affinity chromatography purification to obtain the TF purified protein. The purified TF was assayed for concentration by a protein quantitative determination kit using BCA method.

The preparation process comprises the following steps: the method comprises two parts, namely obtaining TF and constructing a PT kit. TF is obtained by amplification fermentation culture of glycerol bacteria, obtaining of protein supernatant, purification and quantification of protein. The construction of the PT kit mainly comprises the processes of lipidation process, sensitivity adjustment, performance index determination and tracing to international standard products. The detailed implementation scheme is as follows:

(1) and (3) amplification and fermentation of the strain, namely taking out a glycerol strain from a refrigerator at the temperature of-80 ℃, inoculating the glycerol strain to an LB culture medium in a super clean workbench at the evening of the day, transferring the glycerol strain to a small bottle for culture, adding kanamycin antibiotic, culturing the kanamycin antibiotic with the concentration of 50mg/L in a shaking table at 37 ℃ and 200rpm overnight for 12-16 hours, transferring the glycerol strain to a large bottle at the morning on the next day, adding the glycerol strain to the large bottle according to the ratio of the bacteria to the culture medium of 1:25, adding the kanamycin antibiotic again, culturing for 3 hours, detecting OD to 0.6-1.0 by using an ultraviolet spectrophotometer, then carrying out IPTG (isopropyl- β -D-thiogalactoside) induction, carrying out IPTG (isopropyl- β -D-thiogalactoside) induction until the final concentration of 0.5mM, expressing the protein at the low temperature of 16 ℃ overnight, and carrying out centrifugal collection on the morning on the third day.

(2) Centrifuging and crushing: and (3) weighing a centrifugal barrel in advance, adding a bacterial liquid, balancing, centrifuging at 8000G for 5min, discarding the supernatant, and weighing again, wherein the difference between the two masses is the weight of the wet bacteria. Adding 25mL of disruption buffer solution into each gram of wet bacteria for resuspension, and disrupting 3 times at high pressure of 800 bar. The disrupted cells were centrifuged at 12000G for 40min to obtain a supernatant. 1% Triton (Triton) was added and stirred overnight, and the disruption process allowed the sample to cool in an ice bath.

(3) Protein purification and preparation of each purification buffer:

disruption buffer: 25mM Tris-HCl (Tris-hydroxymethyl-aminomethane), 150mM NaCl, 25mM imidazole, adjusted to pH 8.0. (Triton-free)

10% (v/v) Triton disruption buffer: dilution with disruption buffer i.e. 1 part of 100% Triton was added to 9 parts of disruption buffer.

Loading buffer solution: diluted with disruption buffer and 1% Triton added.

And (3) an equilibrium buffer: 25mM Hepes (4-hydroxyethylpiperazine ethanesulfonic acid), 150mM NaCl, 25mM imidazole, 0.5% Triton, adjusted to pH 8.0.

Elution buffer: 25mM Hepes, 150mM NaCl, 250mM imidazole, 0.5% Triton, adjusted pH 8.0.

The crushed supernatant stirred overnight was filtered by a suction pump and centrifuged to remove the precipitate. Ready for purification.

(4) Addition of surfactant to the TF protein. To the TF protein before purification a surfactant is added. The structure of the protein can be opened only by adding a surfactant to make the TF recombinant protein show activity, and the surfactant can be TritonX-100 or NP40 (ethyl phenyl polyethylene glycol), and the surfactant is added before the protein is purified, and is maintained in the whole purification process.

Purifying by using a pre-packed nickel column, keeping the flow rate of purification at 5mL/min in each step, firstly balancing the nickel column by using 5 column volume loading buffer solutions, then loading, balancing by using 10 column volume balancing buffer solutions after loading is finished, finally eluting the sample by using 3 column volume elution buffer solutions, and collecting the eluted sample. The protein concentration was diluted to 1mg/mL with the BCA kit for construction of PT kit.

(5) And (4) preparing a PT reagent. The TF protein was diluted to a concentration of 1mg/ml with a purification eluent containing a surfactant. The rabbit cephalin is prepared into 20mg/ml by pure water and is mixed evenly by ultrasonic. Chaps (surfactant) was formulated with pure water to a concentration of 6%. The content ratio of the three is TF: rabbit brain phospholipids: chaps is 3:10:30, and lipidation is carried out for 2h at 37 ℃ to form lipidated substances. And taking out the lipidated substance and adding a buffer solution, wherein the volume ratio of the lipidated substance to the buffer solution is 1: 36. The buffer was 60mM hepes, 4% glycine, 0.015mg/ml polybrene, 16mM calcium chloride, pH 7.4. The optimal mass ratio of TF to rabbit brain phospholipid to chaps is 3:10:30, and the normal quality control second value of PT can be changed by adjusting the ratio.

(6) Sensitivity of PT reagent. The sensitivity of the PT reagent can be adjusted by adjusting the volume ratio of the TF, rabbit brain phospholipids and chaps mixture to the buffer, and as the volume of the mixture increases, the sensitivity increases. FVII sensitivity also shows the same characteristics as sensitivity.

(7) Heparin resistance of PT reagent. The anti-heparin effect is achieved by adding heparin antagonist polybrene. The addition of polybrene extended the normal quality control seconds. The anti-heparin effect increases with the increase of the polybrene amount, and when the polybrene amount is increased to 0.015mg/ml or more, the anti-heparin effect is obvious and does not need to be increased any more, but the polybrene amount cannot be increased infinitely, and when the polybrene concentration exceeds 0.15mg/ml, the second value is abnormally increased.

(8) The ISI is traceable. And the method follows national/international standard of GB/T21415-2008/ISO 17511:2003 'metrology traceability of measurement calibrator and quality control material assignment of in vitro diagnosis medical apparatus biological samples' for metrology traceability. And (3) purchasing a WHO fifth generation thromboplastin reference product RTF/16 and tracing the source of the PT reagent. The ISI value on the Securid semi-automatic coagulator was 1.127.

The invention constructs, expresses and purifies the human gene recombinant tissue factor to obtain the active human gene recombinant tissue factor through the gene recombinant technology, and prepares the liquid PT reagent through the lipidation process. The reagent has small difference between batches, high sensitivity, anti-heparin interference, sensitivity to coagulation seven Factor (FVII), and traceability to international standards.

The raw materials and reagents used in the buffer composition, the purification preparation, the PT detection composition and the PT detection kit for purifying the tissue factor provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples: