Charge repulsion induced single-chain collagen polypeptide probe and preparation method thereof

A technology of collagen polypeptide and chain collagen, which is applied to the preparation method of peptides, preparations for in vivo experiments, chemical instruments and methods, etc., can solve the damage of the tissue to be tested, the loss of collagen targeting binding ability of polypeptide probes, and the impact The accuracy of the test results and other issues can be avoided to avoid the risk of damage, the preparation is simple, and the effect of broad application prospects

Active Publication Date: 2020-04-24
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent CN107266562A discloses a polypeptide probe for identifying diseased collagen. The probe is modified with fluorescent substances on the repeat sequence of GPO or GPP, and it is easy to form a triple helix structure under normal temperature conditions; however, the triple helix structure will lead to polypeptide detection. Needles lose their ability to target collagen
However, high temperature and other pretreatment processes are likely to cause damage to the tissue to be tested, which will affect the accuracy of the test results

Method used

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  • Charge repulsion induced single-chain collagen polypeptide probe and preparation method thereof
  • Charge repulsion induced single-chain collagen polypeptide probe and preparation method thereof
  • Charge repulsion induced single-chain collagen polypeptide probe and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of Collagen Polypeptide Probe F-PCTP-D1-6

[0054] 1. Sequence Design of Collagen Peptide Probe

[0055] The peptide sequence of the collagen peptide probe is respectively designed as: FAM-Ahx-(GPO) 7 -(Asp) 6 -NH 2 、FAM-Ahx-(GPO) 7 -(Asp) 5 -NH 2 、FAM-Ahx-(GPO) 7 -(Asp) 4 -NH 2 、FAM-Ahx-(GPO) 7 -(Asp) 3 -NH 2 、FAM-Ahx-(GPO) 7 -(Asp) 2 -NH 2 、FAM-Ahx-(GPO) 7 -(Asp) 1 -NH 2 .

[0056] 2. Preparation of Collagen Peptide Probes

[0057] (1) Add 100 mg of Rink ammonia resin to a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0058] (2) Remove the N-terminal Fmoc protecting group from 20% piperidine / N,N-dimethylformamide (DMF) solution, and detect the complete removal of the protecting group by color reaction;

[0059] (3) Dissolve the amino acid (4eq), HOBt (4eq) and HBTU (4eq) whose N-terminus is protected by Fmoc in DMF, activate at low temperature for 20 minutes, add DIEA (6eq) dropwise to the s...

Embodiment 2

[0074] Example 2 Preparation of Collagen Polypeptide Probe R-PCTP-D6

[0075] 1. Design of Collagen Peptide Probes

[0076] The designed collagen peptide sequence is 6-ROX-Ahx-(GPO) 7 -(Asp) 6 -NH 2 , wherein 6-ROX is 6-carboxy-X rhodamine; 2. Preparation of collagen polypeptide probe

[0077] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0078] (2) Remove the N-terminal Fmoc protecting group from 20% piperidine / N,N-dimethylformamide (DMF) solution, and detect the complete removal of the protecting group by color reaction;

[0079] (3) Dissolve the amino acid (4eq), HOBt (4eq) and HBTU (4eq) whose N-terminus is protected by Fmoc in DMF, activate at low temperature for 20 minutes, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor Medium, reaction 3hrs;

[0080] (4) After the reaction finishes, the reaction solution is extracted from the reactor, and the resin ...

Embodiment 3

[0093] Example 3 Collagen polypeptide probe F-PCTP-(GDD) 3 preparation of

[0094] 1. Design of Collagen Peptide Probes

[0095]The designed collagen peptide sequence is FAM-Ahx-(GPO) 7 -(Gly-Asp-Asp) 3 -NH 2 , wherein FAM is carboxyfluorescein.

[0096] 2. Preparation of Collagen Peptide Probes

[0097] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0098] (2) Remove the N-terminal Fmoc protecting group from 20% piperidine / N,N-dimethylformamide (DMF) solution, and detect the complete removal of the protecting group by color reaction;

[0099] (3) Dissolve the amino acid (4eq), HOBt (4eq) and HBTU (4eq) whose N-terminus is protected by Fmoc in DMF, activate at low temperature for 20 minutes, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor Medium, reaction 3hrs;

[0100] (4) After the reaction finishes, the reaction solution is extracted from the reactor, a...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a charge repulsion induced single-chain collagen polypeptide probe and a preparation method thereof. The single-chain collagen polypeptide probe provided by the invention contains a repetitive sequence of (Gly-Pro-Hyp)n, (Gly-Pro-Pro)n or (Gly-Hyp-Hyp)n in the middle, a fluorescence signal molecule ismodified on the sequence, and the C end of the sequence contains a plurality of charged amino acids. The novel polypeptide probe provided by the invention does not contain non-natural amino acid andis simple to prepare. The novel polypeptide probe has pH sensitivity, forms a triple helix structure under an acidic condition, can keep a stable single-chain structure under a physiological pH condition, has the capability of specifically binding lesion collagen without any pretreatment, and completely avoids the damage risk of pretreatment to a tissue sample. The novel polypeptide probe providedby the invention can be used for detecting the content of lesion collagen in vitro, and has a wide application prospect in early diagnosis and curative effect evaluation of collagen-related diseasessuch as tumors.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a single-chain collagen polypeptide functional probe induced by charge repulsion and a preparation method thereof. Background technique [0002] As the most abundant protein in mammals, collagen is the main component of extracellular matrix and plays a key role in tissue formation and maintenance of homeostasis. There is a process of collagen remodeling in the normal development and tissue repair of the human body, that is, the triple helix structure of natural collagen will be deformed or degraded under the action of proteolytic enzymes or other external forces. If during the collagen remodeling process, too much collagen is produced , the excess collagen will accumulate in the organs, prone to tissue fibrosis, and eventually lead to serious diseases such as tumors; if the collagen degrades uncontrollably, it will lead to collagen degenerative diseases, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C09K11/02C07K14/78C07K1/20C07K1/06C07K1/04A61K49/00
CPCC09K11/06C07K14/78C09K11/025A61K49/0056C09K2211/1088C09K2211/1007C09K2211/1029Y02P20/55
Inventor 肖建喜蔡向东
Owner LANZHOU UNIVERSITY
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