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A collagen targeting polypeptide probe containing aromatic amino acids, preparation method and application thereof

A probe and peptide sequence technology, applied in the field of collagen detection, can solve problems such as difficulty in accurate quantification, tissue damage, etc., and achieve the effects of reducing difficulty and cost, broad application prospects, and good fluorescence and luminescence performance.

Active Publication Date: 2021-07-20
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The existing peptide probe GPO that targets and binds to diseased collagen has a very stable triple helix structure, and requires pretreatment such as heating to maintain a single-chain state, which has defects such as difficulty in accurate quantification and risk of tissue damage

Method used

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  • A collagen targeting polypeptide probe containing aromatic amino acids, preparation method and application thereof
  • A collagen targeting polypeptide probe containing aromatic amino acids, preparation method and application thereof
  • A collagen targeting polypeptide probe containing aromatic amino acids, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 polypeptide probe

[0043] 1. Design of peptide probes

[0044] The polypeptide probe sequences designed in this embodiment are: FAM-(Gly-Pro-Hyp) 3 -Gly-Tyr-Hyp-(Gly-Pro-Hyp) 4 、FAM-(Gly-Pro-Hyp) 3 -Gly-Phe-Hyp-(Gly-Pro-Hyp) 4 、FAM-(Gly-Pro-Hyp) 3 -Gly-Asp-Hyp-(Gly-Pro-Hyp) 4 、FAM-(Gly-Pro-Hyp) 3 -Gly-Ala-Hyp-(Gly-Pro-Hyp) 4 、FAM-(Gly-Pro-Hyp) 2 -Gly-Tyr-Hyp-Gly-Pro-Hyp-Gly-Tyr-Hyp-(Gly-Pro-Hyp) 3 、FAM-(Gly-Pro-Hyp) 2 -Gly-Ala-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp-(Gly-Pro-Hyp) 3 ;

[0045] Control peptide probe sequence: FAM-Pro 3 -Gly 3 -Hyp 3 -Pro-Gly-Hyp 2 -Pro 2 -Gly 3 -Hyp 3 -Pro 2 -Gly and FAM-(Gly-Pro-Hyp) 3 -Gly-Pro-Hyp-(Gly-Pro-Hyp) 4 , wherein FAM is carboxyfluorescein.

[0046] 2. Solid phase synthesis of peptide sequences

[0047] (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;

[0048] (2) Remove the N-terminal Fmoc protecting group fr...

Embodiment 2

[0065] Example 2 Polypeptide Probe Targeted Binding Ability to Denatured Collagen

[0066] A 1 mg / mL gelatin solution was prepared in 10 mM PBS (pH 7.4) buffer at 70°C, added to a 96-well plate and air-dried. After film formation, block with 100 μL of 1% BSA blocking solution prepared in 10 mM PBS (pH 7.4) for 1 h, discard the solution in the well, wash with PBS 3 times, 3 min each time;

[0067] Take the polypeptide probes FAM-GPO, FAM-GYO, FAM-GFO, FAM-GDO, FAM-GAO, FAM-2GYO, FAM-2GAO and FAM-Control prepared in Example 1 respectively in 10mM PBS (pH7.4) Prepare a 20 μM peptide probe solution in 20 μM; add 70 μL of the peptide probe solution to a 96-well plate, and incubate at 4° C. for 4 h to allow it to bind to the gelatin membrane. Before use, the peptide probe solution was heated at 80 °C for 15 min to fully melt, and then immediately quenched in ice water for 30 s. The well plate was washed 3 times with 400 μL, 10 mM PBS (pH 7.4), 5 min each time. The fluorescence in...

Embodiment 3

[0069] Example 3 Peptide Probes FAM-GYO and FAM-GFO Targeted Binding Specificity to Denatured Collagen

[0070] The evaluation of the binding ability of the polypeptide probe disclosed in the present invention to denatured collagen, pepsin, trypsin and hemoglobin was carried out according to the experimental scheme described in Example 2: the collagen dissolved in 0.5M acetic acid was heated at 70°C for 15min The solution (type I) was denatured, and 1 mg / mL denatured collagen (type I), hemoglobin, pepsin and trypsin solutions were added to a 96-well plate and air-dried. The orifice plate was washed 3 times with 10 mM PBS (pH 7.4), 3 min each time, and the plate was shaken and patted dry. The FAM-GPO, FAM-GYO and FAM-GFO solutions were heated at 80 °C for 15 min before use and immediately quenched in ice water for 30 s. Add 70 μL (20 μM) of the polypeptide probe solution (prepared as in Example 1) on the protein membrane, and incubate at 4° C. for 4 h. After the binding, wash...

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Abstract

The invention belongs to the technical field of collagen detection, and in particular relates to a collagen targeting polypeptide probe containing aromatic amino acids, a preparation method and an application. The polypeptide probe of the present invention will existing GPO polypeptide probe (Gly-Pro-Hyp) n The Pro in the middle position was replaced by Tyr or Phe, and two novel peptide probes FAM‑GYO containing aromatic amino acids were constructed: FAM‑(Gly‑Pro‑Hyp) 3 ‑Gly‑Tyr‑Hyp‑(Gly‑Pro‑Hyp) 4 and FAM‑GFO: FAM‑(Gly‑Pro‑Hyp) 3 ‑Gly‑Phe‑Hyp‑(Gly‑Pro‑Hyp) 4 . The polypeptide probes FAM-GYO and FAM-GFO significantly reduce the stability of the triple helix structure of the existing GPO probes, and maintain the ability to target and bind diseased collagen equivalent to the existing GPO probes, which can specifically Combined with denatured collagen in different tissues of mice, and successfully applied to tissue staining of different disease samples, it has broad application prospects in the early detection and efficacy evaluation of collagen-related diseases such as tumors and fibrosis. At the same time, the polypeptide probes FAM-GYO and FAM-GFO are short in length and do not introduce complex unnatural amino acids, which greatly reduces the difficulty and cost of probe synthesis.

Description

technical field [0001] The invention belongs to the technical field of collagen detection, and in particular relates to a collagen targeting polypeptide probe containing aromatic amino acids, a preparation method and an application thereof. Background technique [0002] Collagen is a group of extracellular matrix proteins with a unique triple helix structure. A major component of the extracellular matrix, collagen is ubiquitous in connective tissues such as skin, bones, tendons and ligaments. It provides an important structural scaffold for tissue development and mediates various cellular behaviors such as cell adhesion, proliferation, migration and differentiation. Therefore, abnormal collagen remodeling is closely related to various key diseases such as cancer, fibrosis and arthritis. Collagen is considered a key component of the tumor microenvironment, which can affect the behavior of tumor cells and increase the stiffness of tumor tissue. A large number of clinical da...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K1/04C07K1/06C07K1/13C07K1/36C07K1/30C07K1/20C09K11/06G01N33/574G01N33/68G01N21/64
CPCC07K14/001C09K11/06C09K2211/1088G01N21/6486G01N33/57407G01N33/57438G01N33/57446G01N33/57484G01N33/6893G01N2333/78G01N2800/085G01N2800/12Y02P20/55
Inventor 肖建喜魏文宇
Owner LANZHOU UNIVERSITY
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