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Broad-spectrum humanized anti-novel coronavirus monoclonal antibody and application thereof

A monoclonal antibody and coronavirus technology, applied in the field of peptides, can solve the problems of failure, Omicron neutralization ability decline, etc., and achieve the effect of good stability and significant broad-spectrum neutralization ability

Pending Publication Date: 2022-04-29
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The neutralization ability of existing monoclonal antibodies against Omicron has been greatly reduced, or even completely ineffective

Method used

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  • Broad-spectrum humanized anti-novel coronavirus monoclonal antibody and application thereof
  • Broad-spectrum humanized anti-novel coronavirus monoclonal antibody and application thereof
  • Broad-spectrum humanized anti-novel coronavirus monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Synthesis, expression, biotinylation and staining of the novel coronavirus RBD probe

[0043] 1.1 According to the data published by Genbank (NC_045512), synthetically carry 6×His-Avi (His-His-His-His-His-His-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu- Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-Asp-Ile-Thr-Asn-Trp-Leu-Trp-Tyr-ILe-Lys-Lys-Lys) tagged RBD full-length gene sequence.

[0044] 1.2 Reconnected into the eukaryotic expression vector pDRVI1.0 (constructed and preserved by the inventor) after EcoRI and EcoRV double digestion, and the clone was selected and sequenced without error.

[0045] 1.3 The two probe plasmids were respectively transfected into 293F cells for expression. After 5-6 days, the culture medium was centrifuged to collect the cell supernatant, and the antigenic protein was purified through a nickel column.

[0046] 1.4 Use the BirA 500 Biotin Protein Ligase Kit (BirA500, Avidity) to biotinylate the probe protein:

[0047] Dissolve 1 mg mol...

Embodiment 2

[0053] Embodiment 2: screening and identification of anti-SARS-COV-2 humanized monoclonal antibody

[0054] 2.1 Prepare cell lysate: 20 μl cell lysate per well, including 0.5 μl RNase removal, 5 μl 5×FirstStrand buffer, 1.25 μL 0.1M DTT, 0.0625 μl Igepal, 13.25 μl water, cover with sealing film, 4°C Store in the refrigerator for later use.

[0055] 2.2 Sample preparation:

[0056] (1) Resuscitation of PBMCs cells from recovered patients after COVID-19 infection: Take out the frozen cell tube from liquid nitrogen, put it in a 37°C water bath quickly, take it out when it melts until there is an ice core, open it in a biological safety cabinet, and slowly Add R10+ Benzonase medium dropwise (5 mL R10+ Benzonase medium is used for 1 branch of cells). Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, suspend the cells with the residual solution, add 10 mL R10, mix well, take 50 μl for cell counting, and centrifuge at 1500 rpm for 10 minutes; adjust the cell concentra...

Embodiment 3

[0106] Embodiment 3 Antibody is to the neutralizing activity determination of SARS-COV-2 pseudovirus

[0107] 3.1 Packaging of pseudovirus: The full-length S protein gene of artificially synthesized SARS-CoV-2 (GenBank: MN908947) was inserted into the pcDNA3.1 expression plasmid to construct pcDNA-SARS-CoV2-S. The pseudovirus mutation site with mutation is introduced on the S gene as shown in Table 11.

[0108] Table 11. Pseudovirus mutation sites

[0109] .

[0110] will be 3×10 6 (3 million) 293T / 17 cells were seeded in T75 cell culture flasks, 5% CO 2 Incubate at 37°C for 20-24 hours. Use Fugene 6 Transfection Reagent (Promega, Cat#E2691) for transfection: 30ug plasmid pcDNA-SARS-CoV2-S was transfected into 293T cells in a T75 culture flask, and 1.05×10 6 The G*ΔG-VSV virus with TCID50 infected 293T, and the culture medium was changed after 8 hours. After 24 hours of transfection, the culture supernatant was collected and filtered to obtain the pseudovirus of the S...

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Abstract

The invention discloses a broad-spectrum humanized monoclonal neutralizing antibody for resisting SARS-COV-2 virus, which is obtained by screening through a single B cell flow sorting-antibody gene amplification pairing expression technology and has a unique CDR region. The SARS-COV-2 mutant strain can specifically bind to SARS-COV-2 and can effectively neutralize'variant strains (SARS-CoV-2 WT, alpha, Beta, Gamma, Delta and Omicron) which are currently defined by the World Health Organization and worthy of being closed ', and especially the IC50 for neutralizing the Omicron variant strain is lower than 0.1 mu g / mL. The present invention also relates to methods of preparation and uses of the set of neutralizing antibodies. The four antibodies provided by the invention can be used for emergency prevention and / or treatment of global epidemics of COVID-19 variant strains, have the characteristics of full humanization, high expression and good stability, and are suitable for industrialization. In addition, the antibody can also be used for preparing an SARS-COV-2 virus detection reagent, finding effective neutralizing epitopes and developing SARS-COV-2 recombinant protein and subunit vaccines.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an antibody. Background technique [0002] The novel coronavirus (SARS Cov-2, also known as 2019-nCov) is a positive-strand RNA virus belonging to the ß genus of the coronavirus family, which encodes four structural proteins: spike (S), envelope (E), membrane (M), nucleocapsid (N), 16 kinds of non-structural proteins and 5-8 kinds of auxiliary proteins. SARS Cov-2 uses the S protein on the surface of the virus and the host cell receptor-angiotensin-converting enzyme II (ACE2) to enter cells. S protein is divided into two functional units according to protein structure and function, namely S1 and S2 protein subunits. S1 can be divided into NTD (N-terminal domain) and RBD (Receptor binding site). The RBD region is about 240 amino acids long and mainly binds to host cell receptors. S2 plays a role in the fusion of virus and cell membranes. According to existing reports, n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
CPCC07K16/10A61P31/14G01N33/577G01N33/56983C07K2317/565C07K2317/56C07K2317/52C07K2317/76C07K2317/92G01N2333/165G01N2469/10
Inventor 邵一鸣朱彪李丹王铮吴南屏郝彦玲
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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