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Single-chain collagen polypeptide functional probe induced by charge repulsion, and preparation method thereof

A technology of collagen peptides and chain collagens, which is applied in the preparation methods of peptides, preparations for in vivo experiments, chemical instruments and methods, etc., and can solve the problem of non-specificity of pathological collagen, weak probe binding ability, sample tissue sealing, etc. problems, to achieve the effect of no biological toxicity, good target recognition ability, and rapid detection

Inactive Publication Date: 2019-08-16
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, targeted peptides and antibodies have been developed for the identification and imaging of collagen. However, these detection methods are not specific to diseased collagen, the sample needs to be blocked by tissue, and the peptide probe needs to be heated or irradiated by ultraviolet light, which may cause abnormalities. Pretreatment of collagen damage, weak probe binding ability, complex operation and other defects

Method used

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  • Single-chain collagen polypeptide functional probe induced by charge repulsion, and preparation method thereof
  • Single-chain collagen polypeptide functional probe induced by charge repulsion, and preparation method thereof
  • Single-chain collagen polypeptide functional probe induced by charge repulsion, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Design of collagen peptide probe

[0040] The designed collagen peptide sequence is 6-Rox-Ahx-(Gly-Pro-Hyp) 7 -(Asp) 6 , wherein 6-Rox is 6-carboxy-X-rhodamine;

[0041] (2) Preparation of collagen peptide probes

[0042] 1. Add 100 mg Rink ammonia resin to a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0043] 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;

[0044]3. Dissolve the amino acid (4eq), HOBt (4eq) and HBTU (4eq) protected by Fmoc at the N-terminus in DMF, activate at low temperature for 20min, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor , response 3 hrs.

[0045] 4. After the reaction, the reaction solution was extracted from the reactor, and the resin was washed 3 times with 5 mL DMF and DCM respectively. The complete condensation of amino ac...

Embodiment 2

[0056] (1) Design of collagen peptide probe

[0057] The designed collagen peptide sequence is FAM-Ahx-(Gly-Pro-Hyp) 7 -(Asp) 6 , wherein FAM is carboxyfluorescein;

[0058] (2) Preparation of collagen peptide probes

[0059] 1. Add 100 mg Rink ammonia resin to a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0060] 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;

[0061] 3. Dissolve the amino acid (4eq) protected by Fmoc at the N-terminal, HOBt (4eq) and HBTU (4eq) in DMF, activate at low temperature for 20min, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor , response 3 hrs.

[0062] 4. After the reaction, the reaction solution was extracted from the reactor, and the resin was washed 3 times with 5 mL DMF and DCM respectively. The complete condensation of amino acids wa...

Embodiment 3

[0069] (1) Design of collagen peptide probe

[0070] The designed collagen peptide sequence is FAM-Ahx-(Gly-Pro-Hyp) 7 -Gly-(Asp) 5 , wherein FAM is carboxyfluorescein;

[0071] (2) Preparation of collagen peptide probes

[0072] 1. Add 100 mg Rink ammonia resin to a reactor with a sieve plate, and use 5 mL of dichloromethane to swell the resin;

[0073] 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;

[0074] 3. Dissolve the amino acid (4eq) protected by Fmoc at the N-terminal, HOBt (4eq) and HBTU (4eq) in DMF, activate at low temperature for 20min, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor , response 3 hrs.

[0075] 4. After the reaction, the reaction solution was extracted from the reactor, and the resin was washed 3 times with 5 mL DMF and DCM respectively. The complete condensation of amino acid...

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PUM

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Abstract

The invention discloses a single-chain collagen polypeptide functional probe induced by charge repulsion, wherein the single-chain collagen polypeptide functional probe contains a plurality of (Gly-Pro-Hyp)n, (Gly-Pro-Pro)n or (Gly-Hyp-Hyp)n repeat sequences and a polypeptide sequence linkage modifying functional substance, wherein the polypeptide sequence comprises a plurality of charged amino acids at the C-terminus. The preparation method comprises: swelling a resin; activating with an amino acid; carrying out an amino acid condensation reaction; carrying out amino deprotection; extending apeptide resin; after synthesizing the collagen polypeptide of a target sequence, adding 20-30% of acetic anhydride to a reactor, and linking a functional substance to the collagen polypeptide; cutting the resin; and separating and purifying the collagen polypeptide. According to the present invention, the single-chain collagen polypeptide functional probe does not need heating, illumination and other pretreatments so as to completely avoid the potential risk of damage on normal tissues; and the single-chain collagen polypeptide functional probe has good targeting recognition ability, good specificity and strong binding ability to lesioned collagen, and has advantages of rapid detection, simple operation, convenient use, good water solubility and no biological toxicity.

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] Target recognition is a key technology for early detection and accurate diagnosis of tumors and other diseases, and it is widely used in the field of biomedicine. A major component of the extracellular matrix and the most abundant protein in the human body, collagen forms a molecular scaffold that provides mechanical strength and structural integrity to connective tissues such as skin, bones, and tendons. Collagen also participates in the regulation of basic life activities such as cell proliferation, differentiation and migration, and is closely related to major diseases such as osteogenesis imperfecta, arthritis, and tumors. Therefore, collagen is a key biomarker for many diseases in the human body, and the ta...

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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