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Preparation method of fibrinogen degradation fragment

A fibrinogen and fragment technology, which is applied in the preparation methods of fibrinogen and peptides, chemical instruments and methods, etc., can solve the problems of difficult purification and separation, imperfect FDP detection, and difficulty in controlling target peptide segments, etc. high concentration effect

Pending Publication Date: 2022-03-04
BEIJING SICCEEDER TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant record in the prior art on the calibration products related to fibrinogen degradation products. The molecular weight of the segment is smaller than that of D-dimer, and there is little difference between each product segment, so it is difficult to purify and separate
At present, there is no technical content about the preparation of fibrinogen degradation fragments X, Y, D and E. Therefore, providing a preparation method of fibrin degradation product fragments can effectively make up for the imperfect detection of FDP in clinical practice. Positive implications for diagnostic testing of early disease

Method used

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  • Preparation method of fibrinogen degradation fragment
  • Preparation method of fibrinogen degradation fragment
  • Preparation method of fibrinogen degradation fragment

Examples

Experimental program
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preparation example Construction

[0085] 3) Preparation of X fragment, Y fragment and D fragment

[0086] Perform SDS-PAGE on the degraded mixed FDP, use 8% separation gel and 5% stacking gel for electrophoresis, 5% stacking gel for electrophoresis with 80V voltage, 8% separation gel for 120V voltage for electrophoresis, electrophoresis for 1h and then stain for 30min , decolorized observations.

[0087] Fragment X, Fragment Y and Fragment D were recovered by protein glue recovery kit, and protein concentration was tested by BCA protein concentration kit.

[0088] 4) Preparation of E fragment

[0089] The degraded mixed FDP was purified by gel filtration chromatography, using GE molecular sieve superdex200 filler for purification, loading 5 mL of sample, flow rate of 1 mL / min, first running 1 column volume with purified water, and 1 column volume with Tris buffer. Equilibrate the column volume, adjust the UV to zero, observe the increase in conductivity, load the sample, collect the eluted sample, run 1 colu...

Embodiment 1

[0093] This embodiment provides a method for preparing fibrinogen degradation fragments X, Y, D, E, comprising the following steps:

[0094] 1) Degradation of fibrinogen

[0095] Add 20 μl of 150 μg / ml plasmin solution to 1 mL of fibrinogen solution with a concentration of 1 mg / mL, place it at 37 ° C for 30 minutes for degradation, add 10 μL of aprotinin solution with a concentration of 1 mg / mL to terminate the fibrinolytic reaction, and obtain a mixture FDP.

[0096] 2) Preparation of X segment, Y segment and D segment

[0097] Perform SDS-PAGE on the degraded mixed FDP, use 8% separation gel and 5% stacking gel for electrophoresis, 5% stacking gel for electrophoresis with 80V voltage, 8% separation gel for 120V voltage for electrophoresis, electrophoresis for 1h and then stain for 30min , decolorized observations.

[0098] Such as figure 1 The SDS-PAGE results shown are 250kD for the X fragment, 150kD for the Y fragment, 100kD for the D fragment, and 50kD for the X fragm...

Embodiment 2

[0110] Embodiment 2 time affects FDP

[0111] The preparation method is the same as that in Example 1, except that 20 μl of 150 μg / ml plasmin solution is added to 1 mL of fibrinogen solution with a concentration of 1 mg / mL, placed at 37° C. for degradation, and successively at 0, 10 min, 20 min, Mixed FDP was obtained at 30 min, 40 min, 60 min, and 90 min, and tested with a fibrin (original) degradation product (FDP) assay kit. The results are shown in Table 5.

[0112] The FDP concentration (μg / ml) that table 5 different degradation time obtains

[0113]

[0114] It can be seen from Table 5 that under the degradation conditions of this example, the degradation time is 30 min, which has the best degradation effect, and the FDP concentrations obtained by degradation under the conditions of 10 min, 20 min, 40 min, and 90 min are all far less than 30 min.

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Abstract

The invention relates to a fibrinogen degradation technology, and records a preparation method of a fibrinogen degradation fragment, the fibrinogen degradation fragment is degraded by adding a plasmin solution into a fibrinogen solution, in the degradation process, the condition that every 1 mg of fibrinogen is matched with 6 IU of plasmin for degradation is met, the degradation time is 30 min, a mixture of fragments X, Y, D and E is obtained, and the fibrinogen degradation fragment is obtained. And separating and purifying to obtain the target fragment of the fibrinogen degradation product. According to the method disclosed by the invention, the target fragments are accurately prepared, and the prepared degradation product peptide fragments have the characteristics of high purity and concentration, can be used for preparing FDP calibrators and subsequent antibody screening sources, and have positive significance in the field of clinical detection.

Description

technical field [0001] The present application relates to fibrinogen degradation technology, in particular to a preparation method of fibrinogen degradation fragments. Background technique [0002] The fibrinolysis system, referred to as the fibrinolysis system, is the most important anticoagulant system in the human body. Protein (pro) and other protein systems. [0003] The fibrinolytic system plays an important role in maintaining the normal permeability of the blood vessel wall, maintaining blood flow and tissue repair. When fibrin and fibrinogen in fibrinase and Ca 2+ Under the action of different fibrin molecules, the α-chains of different fibrin molecules form cross bonds, so that the fibrin is transformed into the final insoluble fibrin polymer, and the formed fibrin overlaps and overlaps with each other and recruits blood cells, making the original sol-like When the blood turns into a gel-like blood clot, the fibrinolytic system activates plasminogen through the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/75C12P21/06C07K1/16C07K1/26G01N33/68C07K16/36
CPCC07K14/75C12P21/06G01N33/6893C07K16/36G01N2333/75G01N2800/226
Inventor 丁重辉李博华王晓
Owner BEIJING SICCEEDER TECH CO LTD
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