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A method for peptide synthesis and simultaneous construction of peptide magnetic nanoprobes using superparamagnetic nanoparticles as a solid phase

A technology for the synthesis of nanoparticles and polypeptides, which is applied in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc., and can solve problems such as difficulty in obtaining peptide sequences.

Inactive Publication Date: 2016-12-28
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many ways to obtain peptides today (such as using bacterial expression, etc.), there are still many peptide sequences that are difficult to obtain (such as sequences that are difficult to express in bacteria)

Method used

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  • A method for peptide synthesis and simultaneous construction of peptide magnetic nanoprobes using superparamagnetic nanoparticles as a solid phase
  • A method for peptide synthesis and simultaneous construction of peptide magnetic nanoprobes using superparamagnetic nanoparticles as a solid phase
  • A method for peptide synthesis and simultaneous construction of peptide magnetic nanoprobes using superparamagnetic nanoparticles as a solid phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 uses superparamagnetic Fe 3 o 4 Nanoparticle-based solid-phase synthesis of RGDGG pentapeptide and simultaneous construction of RGDGG-magnetic nanoprobes

[0076] Schematic such as figure 1 shown.

[0077] 1. Preparation of silica-coated superparamagnetic Fe 3 o 4 Nanoparticles (prepared according to published methods: Monodisperse MFe 2 o 4 (M)Fe,Co,Mn) Nanoparticles, Shouheng Sun, et.al, J.AM.CHEM.SOC.2004,126,273-279. ) and coupled rink amide linker (4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid, purchased from Jill Biochemical Shanghai Co., Ltd.) Silica coating superparamagnetic Fe 3 o 4 Nanoparticle solid phase:

[0078] Silica-coated superparamagnetic Fe 3 O 4 Preparation of Nanoparticle Solid Phase:

[0079] 1) Prepare 100ml of superparamagnetic Fe with a concentration of 0.05mg / ml 3 o 4 Chloroform solution A of nanoparticles;

[0080] 2) Place solution A at 40°C, and concentrate the solution to 10 microliters...

Embodiment 2

[0113] Embodiment 2 uses superparamagnetic Fe 2 o 3 Nanoparticle-based solid-phase synthesis of RGDGG pentapeptide and simultaneous construction of RGDGG-magnetic nanoprobes

[0114] According to the method for embodiment 1, prepare the superparamagnetic Fe of silica coating 2 o 3 Silica coating of nanoparticles and coupling rink amidelinker (4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid, purchased from Jill Biochemical Shanghai Co., Ltd.) superparamagnetic Fe 2 o 3 nanoparticles. Using the two as solid-phase synthesis of RGDGG pentapeptide and synchronous construction of RGDGG-magnetic nanoprobes, the method is the same as in Example 1.

Embodiment 3

[0115] Example 3 In Vitro Cell Sorting Experiment

[0116] The peptide-conjugated magnetic nanoprobe obtained from the solid phase of superparamagnetic nanoparticles coated with silica in step 3b of Example 1 was dissolved in PBS, and the effect of the synthetic peptide on cells was confirmed by in vitro cell sorting experiments.

[0117] method:

[0118] 1) Add the peptide magnetic probe (0.15mg) obtained from the solid phase of superparamagnetic nanoparticles coated with silica into 1ml of Hela cell and HepG2 cell suspension (the cell concentration is 200,000 cells / ml), and place in a rotating Incubate at 4°C for 2 hours in a mixer;

[0119] 2) Place the incubated two groups of cells on a magnet for 5 minutes, discard the solution, and wash three times with PBS;

[0120] 3) The suspension obtained after the treatment in step 2) is observed under a light microscope, and the strength of the polypeptide's effect on the cells can be judged by the number of cells in the field o...

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Abstract

The invention discloses a method for taking super-paramagnetism nanometer particles as a solid phase to perform peptide synthesis and synchronously construct a polypeptide magnetic nanometer probe. The method comprises: preparing silica-cladded super-paramagnetism nanometer particles and rink-amide-linker-coupled silica-cladded super-paramagnetism nanometer particles; respectively taking the two kinds of particles as solid phases to perform peptide synthesis; and adding a K reagent into two groups of the solid phases. Aiming at the silica-cladded super-paramagnetism nanometer particles, the K reagent is capable of removing a side-chain protection group of the synthetic polypeptide but not cutting off the polypeptide, so that the polypeptide magnetic nanometer probe is obtained. Aiming at the rink-amide-linker-coupled silica-cladded super-paramagnetism nanometer particles, the K reagent is capable of removing the side-chain protection group of the synthetic polypeptide and cutting off the polypeptide from the solid phase, so that the synthetic polypeptide is obtained. According to the method, the super-paramagnetism nanometer particles are capable of being rapidly divorced from a reaction system under the effect of an external magnetic field , so that synthesis is greatly facilitated, also polypeptide can be simply precisely synthesized, also the polypeptide magnetic nanometer probe is synchronously constructed for confirming the biological functions of the synthetic peptide in vitro and in vivo, and also the method is applicable to establishment of large-scale polypeptide library and applicable to rapidly screening out functional peptides.

Description

technical field [0001] The present invention relates to a method for solid-phase synthesis of polypeptides, in particular to a method for synthesizing polypeptides using superparamagnetic nanoparticles as a solid phase, and synchronously constructing synthetic polypeptide nano-magnetic probes while synthesizing polypeptides, which can be used in other biological Function detection, the invention belongs to the technical field of polypeptide solid-phase synthesis. Background technique [0002] 1. Solid-phase peptide synthesis technology: [0003] Solid-phase peptide synthesis technology is an important technology in the field of biochemistry today, and the pioneer of this technology is Robert Bruce Merrifield. Although there are many ways to obtain polypeptides (such as using bacterial expression, etc.), there are still many polypeptide sequences that are difficult to obtain (such as sequences that are difficult to express in bacteria). Using peptide synthesis technology ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/06C07K1/04C12N5/09C12N13/00A61K49/14G01N21/00
Inventor 沙印林罗聃
Owner PEKING UNIV
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