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Affinity peptide M1 of novel corona virus main protease and application thereof

A coronavirus, main protease technology, applied in the field of biomedicine, can solve problems such as false positives

Active Publication Date: 2021-05-28
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection method for coronavirus is mainly nucleic acid detection. Due to reasons such as sampling and sample storage, there may be "false positives". In addition, in addition to symptomatic treatment and supportive treatment, there is still a lack of effective specific treatment for coronavirus Therefore, it is urgent to research and develop more rapid and reliable detection methods, as well as anti-coronavirus drugs with clear targets and new structural types, so as to ensure the ability to prevent coronavirus infection and deal with the outbreak of related infectious diseases

Method used

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  • Affinity peptide M1 of novel corona virus main protease and application thereof
  • Affinity peptide M1 of novel corona virus main protease and application thereof
  • Affinity peptide M1 of novel corona virus main protease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084]Example 1 The virus main protease M of SARS-CoV-2 pro Preparation of the affinity peptide

[0085] 1. Experimental materials

[0086] Ph.D.-12 Phage Display Peptide Library Kit was purchased from New England Biolabs, Inc. Interaction kinetics analysis and affinity determinations used ForteBio Octet instruments. The experimental reagents and experimental operation methods were carried out in accordance with the instructions of the product.

[0087] 2. Experimental method

[0088] Obtaining the main virus protease M of SARS-CoV-2 by phage display technology pro Affinity peptides, using M13 phage, pIII display system peptide library, the library includes 10 9 different polypeptide sequences. Repeat the process of "adsorption-elution-amplification" for 3-5 rounds to obtain phages that can bind to the target protein. Extract the DNA of the phage, complete the sequencing, analyze and obtain the polypeptide sequence that can bind to the target protein, and further synthe...

Embodiment 2

[0149] Example 2 Detection of Affinity and Interaction Kinetics between Affinity Peptide and Target Protein

[0150] 1. Experimental method

[0151] Detection of affinity peptides M1, M3, M4 and target protein M by Bio-layer Interferometry (BLI) pro The affinity of the interaction between them, and the kinetic process of the binding and dissociation between the polypeptide and the target protein. Modify the M1 polypeptide at the C-terminal with Biotin (Biotin-GG-GDAH TRPP WSAW-NH 2 ), immobilized on the SA-modified sensor, the treatment of M3 and M4 in the control group was the same as that of M1, and it was plotted against different concentrations of M pro The binding and dissociation kinetic curves during the interaction, the dissociation equilibrium constant K of their interaction is calculated from the binding and dissociation kinetic curves D , the detailed experimental steps are as follows:

[0152] (1) Immerse the biosensor surface-modified with streptavidin (Strept...

Embodiment 3

[0163] Example 3 Verification of the responsiveness of the affinity peptide M1 to the target protein

[0164] 1. Experimental method

[0165] A cysteine ​​is introduced at the C-terminus of the affinity peptide, so that the peptide is bound to the surface of gold nanoparticles through Au-S bonds. Add a series of concentration gradient target proteins to the system, and detect the SPR peak position of the gold nanoparticles when the protein binds to the affinity peptide on the surface of the gold nanoparticles. The detailed experimental steps are as follows:

[0166] (1) Preparation of gold nanoparticles

[0167] Add 150mL sodium citrate solution (2.2mM) into a 250mL round bottom flask, place in a water bath at 90°C, stir magnetically at 400rpm, add 1mL chloroauric acid solution (25mM), react for 30min, and then add 1mL 60mM citric acid Sodium solution and 1mL 25mM chloroauric acid solution, after reacting for 30min, add 1mL 60mM sodium citrate solution and 1mL 25mM chloroau...

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Abstract

The invention discloses a novel affinity peptide M1 of corona virus main protease and application thereof. The affinity peptide M1 of the corona virus is screened out through a phage display technology, the affinity peptide has high affinity with the main protease Mpro of the corona virus SARS-CoV-2 and can be used as a lead compound of an anti-corona virus drug, further experimental verification shows that affinity peptide M1 modified gold nano particles can be used in auxiliary detection of corona virus, and the affinity peptide M1 provided by the invention provides reference for further research and development of related novel vaccines and anti-corona virus drugs for resisting the corona virus, and has important significance for preventing and controlling pandemic of the corona virus.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an affinity peptide M1 of a novel coronavirus main protease and its application. Background technique [0002] Coronaviruses (Coronaviruses, CoVs) are enveloped, non-segmented, single-stranded positive-sense RNA viruses belonging to the order Nidovirales, Coronaviridae, and Orthocoronavirinae. It exists widely and is susceptible to humans and many animals. Its natural hosts include humans and other mammals, such as cattle, pigs, dogs, cats, mice, and bats. Coronaviruses are one of the main pathogenic pathogens that cause respiratory infections. According to genetic and antigenic criteria, coronaviruses can be divided into 4 categories, namely alpha, beta, gamma and delta coronaviruses (Fehr AR, Perlman S.Coronaviruses: an overview of their replication and pathogenesis[J].Coronaviruses,2015:1 -23.), wherein, α-coronavirus and β-coronavirus only infect mammals, and ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K7/06A61K38/10A61K38/08G01N33/569A61P31/14
CPCC07K7/08C07K7/06G01N33/56983A61P31/14A61K38/00G01N2333/165
Inventor 王晨轩于兰兰王若楠许海燕温涛
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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