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Human antibody binding to coronavirus RBD and application of human antibody

A coronavirus and human-derived antibody technology, applied in applications, antiviral agents, viruses/phages, etc., can solve problems such as monoclonal antibodies with no sequence structure

Pending Publication Date: 2021-09-14
深圳市福田区格物智康病原研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] But so far, the monoclonal antibody that blocks the binding of the new coronavirus to the host ACE2 is still in the theoretical research stage, and there is no technical report on the monoclonal antibody with a clear sequence structure and a clear blocking function of the new coronavirus.

Method used

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  • Human antibody binding to coronavirus RBD and application of human antibody
  • Human antibody binding to coronavirus RBD and application of human antibody
  • Human antibody binding to coronavirus RBD and application of human antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Example 1: Recombinant expression of SARS-CoV-2 antigen and host receptor

[0062] The fully synthesized gene S1RBD (Accession: QHD43416.1, 319-541aa) was cloned into eukaryotic transient expression vectors with His tag or mFc tag at the C-terminus by enzyme digestion, and the obtained expression plasmid was transformed into Escherichia coli was amplified, the S1RBD-his and S1RBD-mFc expression plasmids were isolated, and according to the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the plasmids were transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and S1RBD-mFc was purified by ProA affinity chromatography column to obtain S1RBD-mFc protein. S1RBD-his was purified by HisTrap HP affinity chromatography column to obtain S1RBD-his protein. And the obtained recombinant protein was tested for purity by SDS-PAGE ( Figure 1-2 ). The coding nucleic acid sequence of S1...

Embodiment 2

[0065] Example 2: Isolation of SARS-CoV-2 S1 protein RBD-specific memory B cells

[0066] Whole blood samples (AP8, AP23, AP24, AP25, and AP31) of 5 patients in the recovery period of the new crown were collected from the First Affiliated Hospital of Shantou University, and the IgG and IgM antibodies in the serum of the patients were tested using the new crown antibody detection kit (Livzon reagent) Tested. The result is as Figure 5 As shown, all five serum samples were positive for the new crown antibody IgG. B cells were enriched using RosetteSep kit (Cat: 15064, STEMCELL), and on this basis, FITC-labeled S1-RBD-his was used to capture memory B cells specifically bound to the new crown RBD and performed flow cytometry sorting. The result is as Figure 6 As shown, S1-RBD-specific memory B cells (CD3-CD19+CD27+CD38int S1-RBD+) were sorted in 96-well plates for subsequent single B cell cloning.

Embodiment 3

[0067] Embodiment 3: the amplification of human source anti-SARS-CoV-2 RBD antibody sequence

[0068] Use RNA magnetic beads (Nanjing Nuoweizan) to extract the RNA of a single B cell, and reverse transcribe it into cDNA. The specific method is as follows:

[0069] 1. Aliquot 5 μl of Catch Buffer B (TCL+1% β-ME) into each well to sort single memory B cells.

[0070] 2. Stick the film and centrifuge at 2000rpm for 1min.

[0071] 3. Add 10 μl HO to each well 2 O and 33μl Beads, pipette and mix well, and act at room temperature for 10min.

[0072] 4. Place on a magnetic stand, room temperature for 5 minutes, discard the supernatant.

[0073] 5. Rinse the magnetic beads with 80% ethanol freshly prepared in 200 μl nuclease-free water, room temperature for 30 seconds, and discard the supernatant.

[0074] 6. Repeat rinsing once, discard the supernatant, and air dry for 3 minutes.

[0075] 7. Remove the magnetic stand, add 12 μl Mix 1 to each well, blow and aspirate 5 times, and ...

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Abstract

The invention provides a human antibody specifically binding to a human coronavirus spike protein receptor binding domain (RBD). Coronavirus RBD-specific memory B cells are isolated from peripheral blood mononuclear cells (PBMCs) of a 5-bit novel coronavirus infected rehabilitation person, amplification is performed to obtain a light and heavy chain variable region sequence of 49 antibodies, and transient expression analysis is performed on 34 of the 49 antibodies, wherein 15 of the antibodies are capable of specifically binding to the coronavirus RBD, and at least 3 of the antibodies are capable of blocking the binding of the coronavirus RBD to the receptor ACE2.

Description

technical field [0001] The present invention belongs to the field of antibody engineering, specifically relates to a monoclonal antibody against coronavirus and its application, in particular to a human monoclonal antibody combined with the receptor binding domain RBD of coronavirus spike protein, its preparation method and its application. Background technique [0002] The 2019 novel coronavirus (2019-nCoV), discovered due to cases of viral pneumonia in 2019, was named by the World Health Organization on January 12, 2020. The virus was subsequently named SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV), and the disease it caused was named COVID-19 by the World Health Organization. [0003] Coronaviruses are a large family of viruses known to cause colds as well as more serious illnesses such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has never been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/24C07K2317/92C12N2800/107A61K2039/505
Inventor 管轶桂勋郑作宜王双管静王荣娟陈佩雯焦莎莎李利峰张锦超朱华晨刘大涛
Owner 深圳市福田区格物智康病原研究所
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