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Affinity peptide for tissue-type plasminogen activator and fragment of activator, and application thereof

A technology of plasminogen and activator, which is applied in the field of affinity peptides, can solve the problems of lack of effective methods for separating and purifying tissue-type plasminogen activator, and achieve good separation and purification effect, less immune reaction of the body, strong affinity Harmonious effect

Inactive Publication Date: 2011-01-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the reports in my country involve the study of tissue-type plasminogen activator mutants, and there is a lack of effective methods for isolating and purifying tissue-type plasminogen activator

Method used

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  • Affinity peptide for tissue-type plasminogen activator and fragment of activator, and application thereof
  • Affinity peptide for tissue-type plasminogen activator and fragment of activator, and application thereof
  • Affinity peptide for tissue-type plasminogen activator and fragment of activator, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Design of tissue-type plasminogen activator and its fragment affinity peptide ligand

[0021] The affinity peptide of the invention adopts the method of molecular dynamics and is designed based on the action sites of tissue-type plasminogen activator and plasminogen activator inhibitor.

[0022] The three-dimensional structures of tissue-type plasminogen activator and plasminogen activator inhibitor were taken from the Protein Data Bank (pdb). Select the B chain of plasminogen activator inhibitor, its residue sequence is as follows: Met-Ala-Pro-Glu-Glu-Ile-Ile-Met-Asp-Arg-Pro-Phe-Leu-Phe-Val -Val-Arg-His-Asn-Pro-Thr-Gly-Thr-Val-Leu-Phe-Met-Gly-Gln-Val-Met-Glu-Pro, using the thirty-three tripeptide as the affinity peptide, at room temperature Molecular dynamics simulation of tissue-type plasminogen activator at least 4ns in water and water environment, during which the affinity peptide and tissue-type plasminogen activator are allowed to freely adsorb, and th...

Embodiment 2

[0027] Example 2: Analysis of affinity peptides of tissue-type plasminogen activator and its fragments and their interaction energy

[0028] Using the method described in Example 1, with the threonine mutation system (i.e. respectively X 2 x 5 x 6 site mutation to threonine) as an example, using the formula (1) to calculate the interaction energy between each affinity peptide and tissue-type plasminogen activator, the results are shown in Table 1.

[0029] E. int =E P+L -E P -E L (1)

[0030] In formula (1), E int Indicates the interaction energy between tissue-type plasminogen activator and affinity peptide, E P+L Indicates the total potential energy of tissue plasminogen activator and affinity peptide, E P Indicates the potential energy of tissue plasminogen activator, E L Indicates the potential energy of the affinity peptide.

[0031] Table 1 Analysis of results of tissue plasminogen activator and different affinity peptide ligands

[0032]

[0033] It c...

Embodiment 3

[0038] Example 3: Affinity peptide ligands of tissue-type plasminogen activator and its fragments are linked to a solid phase carrier

[0039] The polyvinyl alcohol hydrogel system is formed by cross-linking six polyvinyl alcohols, each chain is composed of 40 polyvinyl alcohol monomers, and each chain is connected with 5 structures as shown in SEQ ID Nos.1 For the affinity peptide, carry out a molecular dynamics simulation of at least 4ns, and use the formula (1) to calculate the interaction energy between the affinity peptide and tissue-type plasminogen activator after immobilization, and the interaction energy between the affinity peptide and tissue without immobilization The interaction energy of plasminogen activators is comparable. It can be known that the immobilized affinity peptide has good affinity and can be used for separation and purification or detection of tissue plasminogen activator or its fragments.

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Abstract

The invention discloses an affinity peptide for a tissue-type plasminogen activator and a fragment of the activator, and application thereof, and belongs to the technical field of biology. The structure of the affinity peptide is shown as X1X2X3X4X5X6X7, wherein X1 is alanine or glycine, X2 is proline or threonine, X3 and X4 are both glutamic acid, X5 is isoleucine, leucine or valine, X6 is methionine, and X7 is aspartic acid; and an amino acid residue is arranged between the X5 and the X6 preferably and is the isoleucine, leucine or valine. The affinity peptide can be used for separating and purifying or detecting the tissue-type plasminogen activator and the fragment thereof when fixed on a solid-phase support, and has the advantages of high affinity, safety and stability, good application prospect and the like.

Description

technical field [0001] The invention discloses an affinity peptide which can be used for separating and purifying tissue-type plasminogen activator and its fragment, and belongs to the field of biotechnology. Background technique [0002] Tissue plasminogen activator is an important serine protease related to fibrinolysis in the fibrinolytic system. It can activate plasminogen into fibrinolytic enzyme, that is, plasmin, and the activated plasmin can catalyze the dissolution of fibrin and dissolve the thrombus. Because of its specificity and endogenous nature, it is widely used in the clinical treatment of acute thromboembolic diseases. At present, the mutant of tissue plasminogen activator (Val4-Cys261) has been obtained by applying the method of genetic engineering, and the research on how to obtain low-cost and safe tissue plasminogen activator has a good application prospect. [0003] Affinity chromatography is a chromatographic method that uses the specific affinity be...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/22C12N9/50G01N33/50
Inventor 吴韬张锦王琦
Owner ZHEJIANG UNIV
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