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Neutralizing antibody against novel coronavirus sars-cov-2 and its application

A sars-cov-2 and coronavirus technology, applied in antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve problems such as affinity differences

Active Publication Date: 2021-02-19
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subtle differences in the contact interface between the two RBDs and ACE2 lead to differences in the affinity of SARS-Cov-1 and SARS-Cov-2 to ACE2, which may benefit follow-up studies
[0005] Currently, there is no SARS-CoV-2 virus-specific vaccine and neutralizing antibody for clinical treatment

Method used

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  • Neutralizing antibody against novel coronavirus sars-cov-2 and its application
  • Neutralizing antibody against novel coronavirus sars-cov-2 and its application
  • Neutralizing antibody against novel coronavirus sars-cov-2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Screening of fully human antibodies targeting SARS-Cov-2-RBD

[0038] Using phage display technology, with SARS-Cov-2-RBD-his protein as positive antigen, SARS-Cov-1-RBD-hFc as negative antigen, in Tomlinson I&J phage library (Genservice Ltd., Cambridge, UK, library size 1.47x10 8 ) for differential screening.

[0039] Coat immunoplates with 50 μg / ml SARS-Cov-2-RBD his antigen and SARS-Cov-1-RBD hFc antigen at 4°C overnight; block with PBS solution containing 5% skimmed milk powder and 0.1% Tween-20 at room temperature Immunoplate for 1 hour; phage library at 10 12 Mix pfu with 10% skimmed milk powder PBS solution 1:1, incubate at room temperature for 2 hours, add the blocked SARS-Cov-1-RBD hFc antigen immunoplate (100 μl / well), incubate at room temperature for 1 hour, and perform negative antigen pre-adsorption ; After pre-adsorption, the supernatant was transferred to the blocked SARS-Cov-2-RBD his antigen immunoplate (100 μl / well), and incubated at room...

Embodiment 2

[0050] Example 2, Antigen-specific analysis of antibodies

[0051] In this embodiment, ELISA is used to detect the combination of the 4A3 phage of Example 1 and the SARS-Cov-2-RBD protein.

[0052] The specific process is: use 5 μg / ml of SARS-Cov-1-RBD-hFc and SARS-Cov-2-RBD-hFc to coat the immune plate overnight at 4°C; use 3% skimmed milk powder, 0.05% Tween-20 The PBS solution of 0.05% Tween-20 was used to block the immune plate for 1 hour at room temperature; the 4A3 phage was added to the blocked immune plate (50 μl / well), and incubated for 1 hour at room temperature; the immune plate was washed 3 times with 0.05% Tween-20 PBS solution (340ul / well ); mix HRP / Anti-M13 Monoclonal conjugate with a PBS solution containing 5% skimmed milk powder and 0.05% Tween-20 at a ratio of 1:4000, add to the washed immunoplate (50 μl / well), and incubate at room temperature for 1 hour; Wash the immunoplate 5 times with 0.05% Tween-20 in PBS solution; add TMB chromogenic solution to the im...

Embodiment 3

[0054] Example 3, Expression and Purification of Antibodies

[0055] The heavy chain variable region sequence and the light chain variable region sequence of the 4A3 scFv sequence were inserted into pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk vectors (Invivogen, San Diego, CA), respectively, to construct human IgG1 expression plasmids, such as image 3 , Figure 4 shown. After co-transfection of the above plasmids in 293T cells, the supernatant was collected and purified with a protein A-Agarose separation column, and the purity of the antibody was detected by SDS-PAGE, as shown in Figure 5 shown.

[0056] The specific process is as follows: the heavy chain variable region sequence and the light chain variable region sequence of the 4A3 scFv sequence were inserted into pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invivogen, San Diego, CA), respectively, to construct an adult IgG expression plasmid. 5 million HEK293T cells were planted in a cell culture dish with DMEM medium supplemented with ...

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Abstract

The invention relates to neutralizing antibodies against novel coronavirus SARS-Cov-2 and applications thereof. The antibody has at least one of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3. The antibody can be used to prepare diagnostic reagents or diagnostic kits, medicines or pharmaceutical compositions for detecting, preventing and treating COVID‑19. The present invention uses phage display technology to target SARS-Cov-2-RBD and SARS-Cov-1-RBD for differential antibody screening, and obtains neutralizing antibodies against novel coronavirus SARS-Cov-2, which can block SARS The combination of ‑Cov‑2‑RBD and ACE2-positive cells has a significant virus-neutralizing effect on SARS‑Cov‑2 pseudoviruses, providing an effective candidate antibody drug for the prevention and treatment of COVID‑19.

Description

technical field [0001] The invention relates to a neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof, belonging to the technical field of biomedicine. Background technique [0002] The 2019 Novel Coronavirus Pneumonia (COVID-19) is caused by the infection of Novel Coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). More than 2 million people have been infected worldwide, with a fatality rate of over 6% ( https: / / covid19.who.int / ), is a major infectious disease that endangers human health. SARS-CoV-2 is a positive-sense single-stranded RNA virus and belongs to the β-type coronavirus [1] , with a nucleic acid identity of 79.5% to SARS-Cov-1 [2] , and its natural host is unknown. Both SARS-CoV-2 and SARS-Cov-1 infect host cells by binding to angiotensin-converting enzyme 2 (ACE2) on the host cell membrane [3] . [0003] ACE2 is an angiotensin-converting enzyme whose substrates are type I and type II angiotensin. Unde...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/10C07K2317/33C07K2317/565C07K2317/76C07K2317/92G01N33/569G01N2333/165G01N2469/10
Inventor 高威刘晓宇高芳苟黎明
Owner NANJING MEDICAL UNIV
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