Anti-coronavirus antibody and application thereof

A technology of antibodies and variants, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problems of weak neutralization activity and low target affinity, and achieve excellent neutralization activity, excellent blocking effect, high The effect of binding ability

Active Publication Date: 2021-10-08
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these antibodies are obtained quickly, they often exhibit relatively low target affinity and relatively weak neutralizing activity

Method used

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  • Anti-coronavirus antibody and application thereof
  • Anti-coronavirus antibody and application thereof
  • Anti-coronavirus antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0174] Example 1 Preliminary acquisition of candidate antibodies capable of efficiently binding to the new crown RBD protein and the new crown S protein

[0175] Use the RBD domain (receptor binding domain) in the S protein of the 2019 novel coronavirus (SARS-CoV2-2019) to immunize wild-type (BALB / c, a total of 5) or humanized (H2L2, a total of 10) Mice, and the splenocytes and bone marrow cells of the mice whose plasma reacted positively to the antigen were selected and enriched with Miltenyi (Miltenyi Biotec, #130-092-530) and Stem Cell kit (Stemcell, #18957) Antibody-expressing plasma cells (plasma B cells). Then through Beacon (Berkeley Lights, Optofluidic System) initially investigated the binding of single B cell culture supernatant to the new crown RBD protein, and derived the positive cells that the culture supernatant can effectively bind the new crown RBD, and then amplified the variable region of the antibody in a single B cell by molecular cloning sequence, and ...

Embodiment 2

[0180] Example 2 Candidate positive antibodies were transiently expressed and purified using HEK293 cells

[0181] The mammalian expression vectors of the positive antibody strains screened in Example 1 were transfected into HEK293F suspension cells (Gibco, #R79007) using PEI (SIGMA, #24885), and cultured in the culture conditions for 7 days. Culture conditions: 37°C, 5% carbon dioxide, 125rpm. After the culture supernatant was harvested, it was purified by affinity chromatography using protein A (AmMag Protein A Magnetic Beads, Genscript, L00695) purification filler. The purity of the obtained protein was preliminarily checked by SDS-PAGE (SurePAGE, Bis-Tris, 10x8, 4-12%, 12wells, Genscript, M00653).

[0182] The expression of all candidate proteins in HEK293 transient expression system was between about 10mg / L-100mg / L. And the SDS-PAGE results of these candidate antibodies all showed that the purity of the obtained antibodies was >90%.

[0183] The above transient express...

Embodiment 3

[0184] The ELISA binding activity investigation of embodiment 3 candidate positive antibody

[0185] Use the positive candidate antibody obtained in the previous step to detect its binding activity to the new crown S protein by ELISA

[0186] Method description: S protein SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His Tag), (Sino Biological, #40591-V08H) and 2019-nCoV Spike Protein (S1 Subunit, His Tag) (Genscript, Batch number: P9FE001), coat the immunoplate overnight at 4°C, add serially diluted candidate antibodies after blocking (selected concentration for serial dilution, such as 15 μg / mL starting, 10-fold serial dilution, to 0.000015 μg / mL, a total of 7 concentrations ), react at room temperature for 1 hour, and then add the secondary antibody anti-Human IgG (Fc) HRP (Jackson Immuno Research Labs, #109-035-098) to react for 1 hour. The TMB color development kit (Chaozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003) was used for color development. After the reac...

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PUM

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Abstract

The invention discloses an anti-coronavirus antibody and application thereof. The coronavirus is a novel coronavirus, and particularly relates to an antibody specifically combined with a novel coronavirus S-RBD protein and an antigen binding fragment of the antibody. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. Sequences of all the CDRs and variable regions are shown in the invention. The antibody or the antigen binding fragment thereof provided by the invention has relatively high binding capacity with a new coronavirus, and also has relatively excellent neutralizing activity ( IC50 values of the neutralizing activity are all lower than a [mu]g / mL level), a relatively good mutant virus coverage spectrum and various binding epitopes.

Description

[0001] This application claims the priority of Chinese patent application 2020107970201 with a filing date of 2020 / 8 / 10. This application cites the full text of the above-mentioned Chinese patent application. technical field [0002] The invention belongs to the field of biomedicine, in particular to an anti-coronavirus antibody and its application, and the coronavirus is a novel coronavirus, in particular to an antibody specifically binding to the novel coronavirus antigen S-RBD protein and its antigen binding fragment. Background technique [0003] Novel coronavirus infection (the disease caused by the novel coronavirus is officially named by the World Health Organization as COVID-19, and the novel coronavirus as SARS-CoV-2) has caused enormous damage to public health and the economy around the world. It spread rapidly and became a global pandemic of unprecedented severity. It has wreaked havoc on the global economy and healthcare systems, and the effects are likely to b...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569A61K39/395A61P31/14
CPCC07K16/10G01N33/56983A61P31/14C07K2317/565C07K2317/56C07K2317/92G01N2333/165G01N2469/10C07K2317/76A61K2039/505
Inventor 郭宇饶子和刘礼乐邬俊张宏恺汪皛皛付丹聂翠缴鹏卢忠华
Owner NANKAI UNIV
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