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Polypeptide probe for targeted recognition of denatured collagen and detection method thereof

A technology of collagen and targeted recognition, which is applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of increasing the torsion angle of collagen triple helix, unfavorable triple helix structure formation, and triple helix instability. Achieve the effects of good biocompatibility, good targeting ability, and convenient detection

Active Publication Date: 2020-06-02
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the collagen characteristic sequence Gly-X-Y, there are generally the following problems in the setting of Hyp: ① Since Hyp also has a hydroxyl group in addition to the pyrrole ring of Pro, it is not necessary to synthesize it due to the existence of steric hindrance. Easy to synthesize; ②In X position, with C γ The Pro residue of -endo usually stabilizes the triple helix, while the C γ The Pro residue of the -exo fold destabilizes the triple helix while Hyp prefers C γ -exo loop folds, so it will excessively increase the torsion angle of the X position of the collagen triple helix, which is not conducive to the formation of the triple helix structure; ③ GOO (Gly-Hyp-Hyp) sequence is almost absent in the type I collagen chain and does not belong to For the composition sequence of natural collagen, those skilled in the art generally choose natural amino acid sequences as probes instead of non-natural sequences for probe design

Method used

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  • Polypeptide probe for targeted recognition of denatured collagen and detection method thereof
  • Polypeptide probe for targeted recognition of denatured collagen and detection method thereof
  • Polypeptide probe for targeted recognition of denatured collagen and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 polypeptide probe FAM-G (OOG) 8 preparation of

[0046] The designed peptide sequence is FAM-Gly-(Hyp-Hyp-Gly) 8 , wherein FAM is carboxyfluorescein.

[0047] Probe preparation steps:

[0048] (1) Add 100mg Rink ammonia resin into a reactor with a sieve plate, use 5mL dichloromethane to swell the resin;

[0049] (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;

[0050] (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 , the reaction time is 3hrs.

[0051] (4) After the reaction was finished, the reaction solution was extracted from the reactor, and the resin was washed three times with 5 mL of DMF and DCM respectively. The complete condensation of...

Embodiment 2

[0055] Embodiment 2 polypeptide probe FAM-G (OOG) 10 preparation of

[0056] The designed peptide sequence is FAM-Gly-(Hyp-Hyp-Gly) 10 , wherein FAM is carboxyfluorescein.

[0057] Probe preparation steps:

[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 , the reaction time is 3hrs.

[0061] (4) After the reaction was finished, the reaction solution was extracted from the reactor, and the resin was washed three times with 5 mL of DMF and DCM respectively. The complete condensation ...

Embodiment 3

[0095] Embodiment 3 polypeptide probe FAM-G (OOG) 8 Determination of thermal change temperature curve of

[0096] Get the polypeptide probe FAM-G (OOG) that embodiment 1 prepares 8 And configure it into a 300μM polypeptide probe solution, use circular dichroism to characterize the intensity of the polypeptide probe at 225nm, the temperature of the polypeptide probe solution is increased from 4°C to 75°C, and the heating rate is 1.0°C / min, and each temperature After equilibrating for 2min, measure the thermal change temperature curve of the polypeptide probe under different pH conditions ( figure 1 Shown in a), and according to the curve to make the first derivative of the temperature and normalize to get the normalized curve ( figure 1 shown in b).

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Abstract

The invention discloses a polypeptide probe for targeted recognition of denatured collagen and a detection method thereof. The polypeptide probe comprises a targeting polypeptide sequence and a luminescent substance X for modifying the N end of the targeting polypeptide, wherein the targeting polypeptide sequence comprises (Hyp-Hyp-Gly)n. The novel polypeptide probe designed by the invention has good binding capacity to denatured collagen, good biocompatibility and strong specificity, and is simple in synthesis process and convenient in operation process. The probe provides a novel potential detection method for molecular-level diagnosis of important diseases such as cancer, cardiovascular diseases and organ fibrosis.

Description

technical field [0001] The invention relates to a polypeptide probe for targeted recognition of denatured collagen and a detection method thereof, belonging to the technical field of biological detection. Background technique [0002] Collagen is the most abundant protein in the human body and is the main component of the extracellular matrix. It forms a molecular scaffold that provides structural integrity and mechanical strength for connective tissues such as skin, bones, and tendons. Collagen has a very special amino acid sequence, which requires a glycine in every three amino acids, forming a characteristic repeated sequence of Gly-X-Y. Collagen in normal connective tissue presents a unique triple helical structure, and the three peptide chains are precisely packed together due to the interaction of hydrogen bonds between the chains. Under many pathological conditions such as tumors and arthritis, the triple helical structure of collagen molecules will be destroyed by p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07K7/08C07K14/00C07K1/06C07K1/04
CPCC07K7/08C07K14/00G01N33/68Y02P20/55
Inventor 肖建喜魏文宇
Owner LANZHOU UNIVERSITY
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