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Oligopeptide for resisting novel coronavirus infection and preparation method and application thereof

A coronavirus and short peptide technology, which is applied in the field of short peptides against novel coronavirus infection and their preparation, can solve problems such as drug shortage and limited therapeutic effect, and achieve the effect of simple preparation and good clinical application prospects.

Active Publication Date: 2021-03-02
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of this application is to provide an anti-new coronavirus polypeptide inhibitor that is convenient for artificial synthesis and industrial production, so as to solve the shortage of existing drugs and the therapeutic effect limited defects

Method used

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  • Oligopeptide for resisting novel coronavirus infection and preparation method and application thereof
  • Oligopeptide for resisting novel coronavirus infection and preparation method and application thereof
  • Oligopeptide for resisting novel coronavirus infection and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 prepares GK-7 short peptide

[0049] 1) Resin activation: Take 1g of 2-Cl(Trt)-Cl resin (Kanglong Bio, Changzhou) into the reactor, soak it with industrial pure dichloromethane (DCM, Tianquan Chemical, Chengdu) until it swells, and industrial pure Wash with dimethylformamide (DMF, Yushuo Chemical, Shandong) for 1 min; then wash with industrial pure DCM twice, each time for 1-2 min;

[0050] 2) Linkage of the first amino acid: Add 0.4mmol N-fluorenylmethoxycarbonyl-L-isoleucine (Gill Biochemical, Shanghai) and 16mL industrial pure DCM, 1mL analytical pure N,N- Diisopropylethylamine (DIEA, Aladdin Biochemical, Shanghai), reaction 90-120min;

[0051] 3) Blocking of resin unbound sites: add 0.5-0.7mL of analytical grade methanol (Aladdin Biochemical) and 1-1.5mL of industrial pure DCM, after blocking reaction for 20-30min, wash with industrial pure DMF 2-3 times, each time 1-2min;

[0052] 4) Removal of amino acid Fmoc group: add 8-10mL piperidine remover wi...

Embodiment 2

[0062] Example 2 GK-7 short peptide inhibits new coronavirus S1 protein immunoblotting experiment

[0063] (1) Cell culture: human alveolar epithelial cell line A549 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were incubated at 37°C in 5% CO in DMEM cell culture medium (Gibco) containing 10% fetal bovine serum (Gibco, ThermoFisher Scientific, Shanghai). 2 cultivated under conditions.

[0064] (2) Cell inoculation: the culture medium was discarded, rinsed twice with sterilized PBS, and 1 mL of 0.25% trypsin (Gibco) was added to digest the cells until detachment. Add 5mL of culture medium to terminate the trypsin reaction, and mix well by pipetting. Centrifuge at room temperature, 1000rpm, 3min, discard the supernatant, and remove cell debris. Resuspend the cells in 5 mL medium and count the cell concentration. Cells were seeded into a sterilized 6-well plate, 1×10 per well 6 CFU. 37°C, 5% CO 2 Cells were grown to adherent condition...

Embodiment 3

[0078] Example 3 GK-7 short peptide inhibits novel coronavirus pseudovirus infection experiment

[0079] (1) Culture and inoculate the human embryonic kidney cell line HEK-293T-hACE2 (Yunzhou Biology, Guangzhou) that highly expresses virus receptors according to the steps in Example 1. Cells were seeded into 96-well plates at a density of 1×10 4 CFU / well. 37°C, 5% CO 2 Cells were grown to adherent conditions.

[0080] (2) Discard the medium, add serum-free medium and GK-7, and the final concentration of the polypeptide is 100, 75, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μg / mL. Add the new coronavirus S protein pseudovirus (Qingke Bio, Beijing) equipped with psPAX2 and luciferase reporter gene, and the titer is 1×10 7 CFU. The negative control group did not contain pseudovirus, and the positive control group did not contain GK-7.

[0081] (3) GK-7 and pseudoviruses were incubated at 37°C and 5% CO 2 Incubate for 12 h under the same conditions, discard the medium, wash...

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Abstract

The invention discloses a GK-7 oligopeptide for resisting novel coronavirus infection. The amino acid sequence of the GK-7 oligopeptide is GKGDFRI. The invention further discloses a preparation methodand application of the GK-7 oligopeptide. The GK-7 oligopeptide is derived from human body protein, contains key residues of a virus-binding cell receptor, and is simple to prepare and good in clinical application prospect.

Description

technical field [0001] This application relates to the technical fields of biopharmaceuticals and polypeptide synthesis, and specifically relates to a short peptide against novel coronavirus infection and its preparation method and application. Background technique [0002] The pneumonia epidemic caused by the new coronavirus seriously threatens human health. The clinical symptoms of patients with new coronary pneumonia are similar to those of the SARS outbreak in 2003. The main manifestations are fever, fatigue, and dry cough. Severe patients have dyspnea one week after the onset, and rapidly develop into acute respiratory distress syndrome, septic shock, dyspnea, and dyspnea. Corrected metabolic acidosis, coagulopathy, and multiple organ failure. [0003] The new coronavirus has a single-stranded positive-strand RNA wrapped in a shell, encoding a spike protein (hereinafter referred to as S protein), an envelope protein, a membrane protein, and a nucleoprotein. The S prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48A61K38/48A61P31/14
CPCC12N9/485A61P31/14C12Y304/17023A61K38/00Y02P20/55Y02A50/30
Inventor 王成王军平韩松伶赵高梅陈银
Owner ARMY MEDICAL UNIV
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