Anti-novel coronavirus Spike protein antibody and application thereof

A coronavirus and antibody technology, applied in the fields of genetic engineering and immunology, can solve the problem of lack of vaccines and specific drugs, and achieve the effect of good ability to bind S protein and good specificity

Active Publication Date: 2021-11-23
EXCYTE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Novel coronavirus 2019-nCoV infection and related diseases caused by its infection currently have no corresponding vaccines and specific drugs, so the development of 2019-nCoV diagnosis and treatment drugs has become a top priority

Method used

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  • Anti-novel coronavirus Spike protein antibody and application thereof
  • Anti-novel coronavirus Spike protein antibody and application thereof
  • Anti-novel coronavirus Spike protein antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0070] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Example 1 Screening Anti-New Coronavirus (2019-nCoV) Spike Protein Antibody from Natural Human Antibody Phage Surface Display Library

[0071]In order to obtain human antibodies specific to the novel coronavirus (2019-nCoV) Spike protein, solid-phase screening was used for panning. First, Spike's RBD protein (mFc tag, Sino biological, Cat. No. 40592-V05H) package was coated. Thaw a human antibody library containing 10 billion phage particles expressing different antibodies. Wash the coated RBD protein target well overni...

Embodiment 2

[0076] Example 2 Antibody function verification

[0077] In order to verify whether the obtained new coronavirus (2019-nCoV) Spike protein antibody clone is combined with the new coronavirus (2019-nCoV) Spike protein antigen and the new coronavirus (2019-nCoV) Spike protein expressed on the cell membrane surface, the new coronavirus (2019 -nCoV) The gene of the Spike protein single chain antibody was cloned into the eukaryotic expression vector pFH. In this vector, the scFv gene is fused with the Fc gene of human IgG to express a protein in the form of scFv-Fc, which can be affinity purified with Potein-A or labeled with (HRP or fluorescein) anti-human Fc antibody detection.

[0078] After obtaining the scFv-Fc protein, the binding of the antibody to the new coronavirus (2019-nCoV) Spike protein and RBD was detected by Octet Blitz, and the specific binding of the new coronavirus (2019-nCoV) Spike protein antibody was confirmed ( Figure 2A-Figure 2E ). CS1 is the monoclonal...

Embodiment 3

[0082] Based on the Cell binding method of FACS analysis, the antibody competes with ACE2 to bind to the cell line ID8 that specifically expresses the 2019-nCoV Spike protein.

[0083] It was determined that the minimum concentration of ACE2-mFC protein that saturates Spike protein in ID8 cells was 0.02 μg / ml.

[0084] 1. Collect ID8 cells: collect 0.5×10 6 cells / tubes.

[0085] 2. Rinse the cells: Rinse the cells once with 1 ml staining buffer (PBS containing 1% w / v BSA), centrifuge at 350×g for 5 min at 4° C., and resuspend with 95 μl staining buffer after centrifugation.

[0086] 3. Antibody binding: Add antibodies CS1-CS8 (0-40 μg / ml) in different concentration gradients, and incubate on ice for 60 minutes.

[0087]4. ACE2-mFC binding: add human ACE2-mFC protein to a concentration of 0.02 μg / ml, and incubate on ice for 30 minutes.

[0088] 5. Rinse the cells: Add 1ml staining buffer to the cell suspension, mix well, centrifuge at 350g at 4°C for 5min, remove the superna...

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Abstract

The invention provides an anti-novel coronavirus Spike protein antibody and an application thereof. The specific antibody against the novel coronavirus S protein is screened from a single-chain antibody library of a non-immune fully human source sequence by utilizing genetic engineering and a phage surface display library technology. The affinity of the antiviral S protein antibody and the virus S protein is between 1 nM and 50 nM, and the antibody has an inhibition effect on the binding of the novel coronavirus S protein and a human receptor ACE2, so that the antiviral S protein antibody has good S protein binding capability and potential neutralization inhibition effect. The invention provides the specific antibody candidate molecule for research and development of diagnostic reagents and antibody drugs for preventing and treating the novel coronavirus (2019-nCoV), treatment of other diseases such as pneumonia caused by the coronavirus, and the like.

Description

technical field [0001] The invention relates to the fields of genetic engineering and immunology, in particular to an anti-new coronavirus Spike protein antibody and its application. Background technique [0002] The novel coronavirus (2019-nCoV) is a new type of coronavirus, which belongs to the β-CoV of the family Coronaviridae of the order Reticuloviridae with SARS-CoV. It is a single-stranded positive-sense RNA virus without segments. The length of each genome is about 30,000 nucleotides. Unlike Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), the novel coronavirus is the seventh in a family of coronaviruses that infects humans. According to the gene sequence homology, the genome of the new coronavirus has 80% similarity with SARS, and the gene sequence of the new coronavirus has 40% similarity with MERS-CoV. [0003] The novel coronavirus 2019-nCoV exhibits a typical coronavirus structure ( Figure 4...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14A61P11/00G01N33/569
CPCC07K16/10A61P31/14A61P11/00G01N33/56983C12N2800/22C07K2317/622C07K2317/92C07K2317/565C07K2317/56A61K2039/505Y02A50/30
Inventor 袁清安孟庆武白丽莉赵立坤李延虎薛晓莹温振国张国良郭大庆张吟秋郑珊珊陈小军杨吉董冉武修竹黎春盈
Owner EXCYTE LLC
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