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Polypeptide, preparation method and application thereof

A kind of use and activity-inhibiting technology, which is applied in the field of polypeptide drugs, can solve the problems of immune response and reduce the enzyme stability of polypeptide drugs, and achieve the effect of good stability

Active Publication Date: 2021-10-29
SHANXI JINBO BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the polypeptide targeting the seven-repeated helix sequence (HR1) of the coronavirus spike protein (S protein) has broad-spectrum anti-coronavirus activity, and the activity is further modified by lipids and polyethylene glycol (PEG) It has been greatly improved, but there are reports in the literature that PEG-modified drugs may cause immune reactions and reduce the enzyme stability of polypeptide drugs

Method used

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  • Polypeptide, preparation method and application thereof
  • Polypeptide, preparation method and application thereof
  • Polypeptide, preparation method and application thereof

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preparation example Construction

[0094] The invention also provides a preparation method of the polypeptide or fusion protein. The method includes introducing the expression vector into host cells, then culturing in a suitable medium, then harvesting or isolating the polypeptide or fusion protein in the supernatant, or in the case of intracellular expression, the host cells can be collected, and then the cells Cleavage is performed to collect these polypeptides or fusion proteins. In the case of a polypeptide or fusion protein, the polypeptide or fusion protein may be linked to a suitable signal peptide. The signal peptide is cleaved or not cleaved after harvest. These techniques are well known to those skilled in the art.

[0095] The polypeptides of the present invention can be prepared in the form of derivatives. For example, polypeptides can be palmitoylated or cholesterol modified. Methods for palmitoylation modification or cholesterol modification are well known. In one embodiment, palmitoylation o...

Embodiment 1

[0115]Embodiment 1: the preparation of SARS-CoV-2 (comprising its mutant), SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, SARSr-CoV-Rs3367, SARSr-CoV-WIV1-CoV pseudovirus

[0116] experimental method:

[0117] 1. 24h before transfection, plate 293T cells in a 10cm tissue culture dish (2×106 / dish); culture with DMEM containing 10% FBS for 12h;

[0118] 2. Replace the cells with pre-warmed fresh DMEM medium (containing 10% FBS) 2 hours before transfection;

[0119] 3. Use two 1.5mL EP tubes for transfection, and add 500 μL of 0.9% NaCl solution to EP tube 1, which contains 20 μg of pcDNA3.1-SARS-2-S plasmid or pcDNA3.1-SARS- S plasmid or pcDNA3.1-MERS-S plasmid or pcDNA3.1-NL63-S plasmid or pcDNA3.1-OC43-S plasmid or pcDNA3.1-WIV1-S plasmid or pcDNA3.1-Rs3367-S plasmid or pcDNA3. 1-SARS-S mutant plasmid and pNL4-3.luc.RE plasmid. Related information can also be found in the literature under, S. # , Yan, L. # , Xu, W. # , Agrawal, A.S., Alsaissi, A., Tseng, C.T.K., Wang, Q., Du,...

Embodiment 2

[0125] Example 2: Inhibition of SARS-CoV-2 S protein-mediated cell-cell fusion experiment (cell-cellfusion)

[0126] experimental method:

[0127] 1. Transfect 293T cells with a plasmid encoding the S protein of SARS-CoV-2 (pAAV-IRES-GFP-SARS-CoV-2-S) and culture for 36-48h to obtain transfected cells as effector cells, Called 293T / 2019 / EGFP cells.

[0128] 2. Digest the 293T / 2019 / EGFP cells with 0.02% EDTA, centrifuge, resuspend the cells in fresh DMEM medium with 10% FBS, and adjust the cell concentration to 2×10 5 1 / mL, 50 μL was taken out and added to various test peptide drugs (50 μL) in serial dilution, and incubated at 37° C. for 30 min. Cells without drugs were used as positive control cells.

[0129] 3. Take 100 μL of the cell / drug mixture and add it to the target cell Huh-7 that has been plated on a 96-well plate. 5%CO 2 , Incubate at 37°C for 2-4 hours, observe and record the fusion of cells with the green fluorescent channel of a fluorescence microscope.

[0...

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Abstract

The invention provides a polypeptide containing an amino acid sequence shown in SEQ ID NO: 1. The invention also provides a derivative of the polypeptide modified by palmitic acid or cholesterol. The polypeptide can inhibit infection of a novel 2019 coronavirus (SARS-CoV-2) and various human coronaviruses, and can also inhibit infection of a type I human immunodeficiency virus (HIV-1). The polypeptide provided by the invention has broad-spectrum antiviral activity and has higher stability than PEG modified polypeptide, and the invention aims to provide good prevention and treatment medicine reserve for SARS-CoV-2 which is still popular at present and a mutant strain of the SARS-CoV-2 as well as SARS-related viruses and human immunodeficiency viruses which are likely to break out in the future.

Description

technical field [0001] The invention relates to the field of polypeptide medicine, in particular to a polypeptide for broad-spectrum inhibition of coronavirus and type I human immunodeficiency virus (HIV-1). Background technique [0002] Type I enveloped viruses such as human immunodeficiency virus (HIV) and highly pathogenic human coronaviruses (HCoV), including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), among others, pose serious threats to global public health and the economy. Therefore, it is an important task to develop effective broad-spectrum anti-enveloped virus drugs to prevent the spread of existing type I enveloped viruses among humans and the possibility of new type I enveloped viruses infecting humans in the future. [0003] In the process of type I enveloped virus infecting target cells through membrane fusion, its main feat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/864C12N5/10A61K38/16A61K45/06A61K47/54A61P31/14A61P31/18
CPCC07K14/005C12N15/86C12N5/0686A61K38/162A61K45/06A61K47/554A61P31/14A61P31/18C12N2770/20022C12N2770/20033C12N2750/14143C12N2800/107C12N2510/02A61K38/00A61K2300/00
Inventor 王茜陆路姜世勃周洁徐巍徐菱
Owner SHANXI JINBO BIO PHARMA CO LTD
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