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Neutralization activity monoclonal antibody of humanized anti-novel coronavirus (SARS-CoV-2)

A coronavirus, sars-cov-2 technology, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problem of time required for clinical application, and achieve the effect of high affinity

Active Publication Date: 2021-01-15
李亚峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some therapeutic drugs have been reported, more clinical practice is still needed to prove the effect. The research and development of related vaccines is also underway, but it will take time before clinical application

Method used

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  • Neutralization activity monoclonal antibody of humanized anti-novel coronavirus (SARS-CoV-2)
  • Neutralization activity monoclonal antibody of humanized anti-novel coronavirus (SARS-CoV-2)
  • Neutralization activity monoclonal antibody of humanized anti-novel coronavirus (SARS-CoV-2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Antigen Preparation

[0088] Cloning, expression and purification of Spike antigen of 2019-nCoV

[0089] The sequence of the Spike protein of the new coronavirus was obtained from NCBI, and after codon optimization, it was constructed into the pcDNA3.1 vector. The vector was obtained by Qiagen's EndoFree Plasmid Kit endotoxin-free plasmid extraction kit to obtain a transfectable plasmid, and lipo3000 was used to transiently transfect 293T The cells were transiently expressed at 37 degrees for 7 days, and the cell expression supernatant was harvested, and then affinity purified using a Ni column to obtain the new coronavirus Spike protein with a purity greater than 80%. The purification results are as follows figure 1 shown.

Embodiment 2

[0090] Example 2 Antibody Separation and Screening

[0091] The prepared Spike antigen-labeled FITC fluorescent dye was purified to obtain S-FITC; peripheral blood lymphocytes were isolated from the peripheral blood of recovered volunteers, and B cells were enriched through the magnetic bead scheme; anti-IgG PE, anti-IgMPerCP- B cells were stained with Cy5.5 and S-FITC, and the IgG+IgM-S+ group new coronavirus S antigen-specific memory B cell subsets were directly sorted into 96 wells that had been added to the cell lysate by flow sorting In a PCR plate, one cell per well.

[0092] Magnetic bead enrichment and sorting single cells into 96-well plates: magnetic bead manufacturer stemcell

[0093] 1. Cell resuscitation: 1 tube each of frozen PBMC cells from recovered patients and PBMC cells from healthy people (cell volume 1*10 7 Cells) were thawed quickly at 37 degrees, and the frozen cells were added to 8ml buffer1 (PBS+2% FBS) preheated at 37 degrees in advance, and centrif...

Embodiment 3

[0109] Example 3 Determination of Antibody Sequence

[0110] By single-cell reverse transcription, the variable region genes of the antibody light chain and heavy chain of a single B cell in each well are transferred.

[0111] Single-cell reverse transcription followed the steps of the vazyme N711 kit to obtain double-stranded cDNA, which was used as a template to extract the light and heavy chain genes of a single B cell antibody. System: 25 μl vazyme P515, 2 μl upstream and downstream primers, 1 μl template cDNA , DDW 20μl, the PCR reaction conditions are 95°C, 3min, 95°C, 15s, 60°C, 15s, 72°C, 30s, 72°C, 7min. After gel identification, the positive rate of antibody gene PCR is above 85%.

[0112] image 3 Part of the result of heavy chain gene amplification, Figure 4 Part of the results for light chain gene amplification. Finally, the variable region sequence of the antibody 23D145 gene derived from recovered volunteers is obtained, as shown in SEQ ID NO.7-8, the CDR s...

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Abstract

The invention provides a novel humanized coronavirus neutralizing active antibody and application thereof. According to the novel humanized coronavirus neutralizing active antibody, the monoclonal antibody with neutralizing activity is separated and screened from the body of a rehabilitation patient infected by the novel coronavirus, has excellent affinity and specificity to the novel coronavirus(SARS-CoV-2), and can effectively block the combination of the novel coronavirus and receptor protein.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to a human-derived novel coronavirus (SARS-CoV-2) neutralizing antibody. Background technique [0002] The new coronavirus is a new type of coronavirus of the genus β, with an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. The whole genome comparison found that the homology between the new coronavirus and the severe acute respiratory syndrome virus (SARS-CoV) reached 70%, and the sequence difference was mainly reflected in the key spike gene (encoding S-protein) that interacts with the host cell. [0003] Usually after infection with the new coronavirus, pneumonia symptoms such as fever, fatigue, and dry cough appear. A small number of patients are accompanied by upper respiratory and gastrointestinal symptoms such as nasal congestion, runny nose, and diarrhea. Severe cases often develop dyspnea one week later, and severe cases rap...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCC07K16/10G01N33/56983G01N2333/165C07K2317/76C07K2317/56
Inventor 李亚峰
Owner 李亚峰
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