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Probe for specifically detecting pathological collagens as well as preparation method and application of probe

A collagen and probe technology, applied in the field of collagen detection, can solve the problems of increasing the cost of probe synthesis, complicating the synthesis process, and affecting the detection results, etc., achieving good fluorescence performance, good single-chain stability, and wide application foreground effect

Active Publication Date: 2020-06-19
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the synthesis of the polypeptide sequence GPO, GPP or GOO is relatively difficult, and the introduction of several charged amino acids into the sequence will further complicate the synthesis process and increase the synthesis cost of the probe.
For another example, Chinese patent CN107530454A discloses a peptide conjugate, which is a dimeric collagen peptide formed by linking two collagen peptides through a linker. However, the structure of the peptide conjugate is complex, and the preparation Easy mismatching in the process leads to large differences in the structure of the obtained peptide conjugates, which affects the detection results

Method used

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  • Probe for specifically detecting pathological collagens as well as preparation method and application of probe
  • Probe for specifically detecting pathological collagens as well as preparation method and application of probe
  • Probe for specifically detecting pathological collagens as well as preparation method and application of probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The polypeptide probes GOP-8, GOP-10 and GOP-12 prepared in the following examples can specifically bind to pathological collagen, and specifically take GOP-10 as an example for tissue fluorescence staining, but the polypeptides of the present invention The probe is not limited to GOP-10, other probes also have good fluorescent staining ability and can specifically bind to diseased collagen. The preparation of embodiment 1 polypeptide probe GOP-4

[0031] 1. Design of peptide probes

[0032] The polypeptide probe sequence designed in this embodiment is: FAM-Ahx-(GOP) 4 -NH 2 , wherein FAM is carboxyfluorescein.

[0033] 2. Solid phase synthesis of peptide sequence Ahx-(GOP) 4

[0034] (1) Add 100mg Rink ammonia resin into a reactor with a sieve plate, use 5mL dichloromethane to swell the resin;

[0035] (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 b...

Embodiment 2

[0042] The preparation of embodiment 2 polypeptide probe GOP-6

[0043] 1. Design of peptide probes

[0044] The peptide probe sequence designed this time is: FAM-Ahx-(GOP) 6 -NH 2 , wherein FAM is carboxyfluorescein.

[0045] 2. Solid phase synthesis of peptide sequence Ahx-(GOP) 6

[0046] (1) Add 100mg Rink ammonia resin into a reactor with a sieve plate, use 5mL dichloromethane to swell the resin;

[0047] (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;

[0048] (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 In, react 3h.

[0049] (4) After the reaction was finished, the reaction solution was extracted from the reactor, and the resin was washed three times w...

Embodiment 3

[0054] The preparation of embodiment 3 polypeptide probe GOP-8

[0055] 1. Design of peptide probes

[0056] The peptide probe sequence designed this time is: FAM-Ahx-(GOP) 8 -NH 2 , wherein FAM is carboxyfluorescein.

[0057] 2. Solid phase synthesis of peptide sequence Ahx-(GOP) 8

[0058] (1) Add 100mg Rink ammonia resin into a reactor with a sieve plate, use 5mL dichloromethane to swell the resin;

[0059] (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;

[0060] (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 In, react 3h.

[0061] (4) After the reaction was finished, the reaction solution was extracted from the reactor, and the resin was washed three times w...

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PUM

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Abstract

The invention belongs to the technical field of detection of collagens, and specifically relates to a polypeptide probe for specifically detecting pathological collagens in tissues as well as a preparation method and application of the polypeptide probe. The polypeptide fluorescence probe disclosed by the invention contains a polypeptide sequence (Gly-Hyp-Pro)n and a signal molecule modifying an Nterminal of the polypeptide sequence (Gly-Hyp-Pro)n. The preparation method of the polypeptide probe is simple and convenient, and the polypeptide probe has good single chain stability and fluorescence luminescence performance; the probe is capable of specifically binding with the pathological collagens and can be used for detecting the content of the pathological collagens in the tissues; and the polypeptide probe can also be taken as a tissue imaging regent for early diagnosis of collagen relevant diseases.

Description

technical field [0001] The invention belongs to the technical field of collagen detection, and in particular relates to a polypeptide probe for specifically detecting pathological collagen in tissues, a preparation method and an application. 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. Studies have shown that when the balance of collagen production and degradation is disrupted, many diseases such as osteoporosis and musculoskeletal tissue damage will be caused. Studies have also found that 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 too much collagen is produced during the collagen remodeling process, the excess...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/13C07K1/04G01N33/68
CPCC07K14/00G01N33/68G01N2333/78G01N33/533G01N33/6887G01N2800/105C07K14/78G01N2800/10
Inventor 肖建喜蔡向东
Owner LANZHOU UNIVERSITY